MXPA99006232A - Method for solubilization and naturation of somatotropins - Google Patents

Abstract

A method for the solubilization and/or naturation of a somatotropin involves contacting a somatotropin with a detergent composition and water under conditions effectiveto obtain a naturated somatotropin, wherein the detergent composition may be a C10, C12, C16 or C18 acyl glutamate, a C10, C14 or C18 alkyl sulfate, an alcohol ethoxy sulfate, lauroyl ethylenediamine-triacetic acid (LEDA), a C10 to C18 linear alkyl benzene sulfonate, diphenyl disulfonate or an acyl amino acid.

Description

METHOD FOR THE SOLUBILIZATION AND RENATURALIZATION OF SOMATOTROPINES This application claims the benefit of the provisional application of E.U.A. Serial No. 60 / 034,808, filed on December 31, 1996.

BACKGROUND OF THE INVENTION Somatotropins are growth hormones that were originally discovered in pituitary gland extracts from several animals. Recombinant DNA technology has allowed the expression of somatotropins as heterologous proteins from different host cells. Said recombinant somatotropins, for example, somatotropins produced in a microorganism, e.g. E. Coli bacteria that have been transformed using recombinant DNA are typically produced by a host cell in a denatured, precipitated state, which had little or no bioactivity at all. The absence of bioactivity is generally attributed to the conformation of the recombinant somatotropin molecule, which lacks formation of disulfide bonds. It is believed that recombinant somatotropins are produced by the host cell in a substantially reduced form (without disulfide bonds), due to a relatively high oxide-reduction potential of host cells, such as the E. coli cell. The majority of recombinant somatotropins, such as bovine somatotropins (bST) and porcine somatotropins (pST), are packaged in the host cell as inclusion bodies, also known as refractile bodies, which are cytoplasmic aggregates containing the recombinant somatotropin and the oligomers. In order to recover the recombinant somatotropin in a bioactive state, the somatotropin, for example, in the form of inclusion bodies, is preferably but not necessarily isolated from the host cell, after which the somatotropin can be solubilized to form somatotropin monomers, which can then be renatured in a bioactive conformation. Then, the renatured somatotropin can be purified to remove impurities like those of other somatotropin species, for example, oligomers such as dimers, and host cell proteins, for example, by ion exchange to precipitate impurities (eg, as it is described in U.S. Patent No. 5,182,369, which is incorporated herein by reference) or other suitable techniques. For example, the patent of E.U.A. No. 4,652,630, Bentle et al., Which is incorporated herein by reference, refers to a method for the solubilization and naturalization of the somatotropin protein of the inclusion bodies using an aqueous solution of urea to solubilize the inclusion bodies that contain the recombinant somatotropin. Bentle et al. Expose the use of urea solutions having varying concentrations from 2.5 to 7.5 M and a pH between 9 and 12 for the solubilization step. Once solubilized, the somatotropin protein can be renatured according to the method of Bentle and others at an alkaline pH. Other methods use detergent to solubilize and renature a somatotropin. For example, European Patent Specification publications Nos. 229,100 and 236,902 (The Upjohn Co.), which are incorporated herein by reference, show a method for converting an insoluble form of somatotropin from a transformed microorganism into a binding conformation. of disulfur? in its natural form by solubilizing and oxidizing the somatotropin in the presence of the detergent. This method uses a sodium dodecyl sulfate detergent (SDS) or a detergent whose formula is: CH3- (CH2) n-CO-NR? -CHR2-COOH, where n is 8 to 20 inclusive; Ri is methyl or ethyl; and R2 is hydrogen, ethyl, methyl, n-propyl or isopropyl. These specifications show below a particular method for solubilizing recombinant bST using an aqueous solution of N-lauroylmethylglycine, which is represented by the formula: CH3- (CH2)? O -CO-NRrCHR2-COOH, in a borate pH regulator of sodium at a concentration of 0.1 to 0.5 M and at a pH of 8 to 10.5. The publications of European Patent Specification Nos. 229,110 and 263,902, state that after solubilization and naturalization, the detergent is removed by an anion exchange resin.

Other publications also describe methods for the solubilization and naturalization of recombinant somatotropins using various detergent and non-detergent compounds, among which the US patents are included. Nos. 4,677,196 and 4,766,224, which also use SDS; the patent of E.U.A. No. 5,023,323, which uses SDS in combination with a chaotropic agent such as urea or guanidine hydrochloride; the patent of E.U.A. No. 5,240,834, which uses sarcosyl (N-lauroyl sarcosine), and the US patent. No. 4,975,529, which utilizes 2-amino-2-methyl-1-propanol. Each of these patents are incorporated herein by reference. Although SDS has been used in several references in the methods of solubilization and naturalization of somatotropin, it is well known that SDS binds relatively strongly to renatured somatotropin, thus making it difficult to extract completely from the solution. somatotropin. There is a need in the art to find more economical and efficient methods to obtain recombinant somatotropins with high relaxation and purity. In particular, there is a need for methods for the solubilization and naturalization of recombinant somatotropin proteins to obtain somatotropin molecules in a bioactive state, preferably using a small amount of readily biodegradable detergent. There is also a need for methods that use detergent that can be easily removed from the renatured somatotropin.

BRIEF DESCRIPTION OF THE INVENTION This invention generally relates to methods for producing biologically active recombinant somatotropins. More particularly, this invention relates to methods for the solubilization and naturalization of recombinant somatotropins comprising the contact of a somatotropin with a detergent composition and water under conditions effective to obtain a renatured somatotropin, wherein the detergent composition contains an acylglutamate. from Cio to C18, a C 1, C or C 18 alkylsulfate, an alkoxylethoxysulfate, a lauroylethylenediaminetriacetic acid (LEDA), a linear alkylbenzene sulphonate of C 1 to 4, diphenyldisulfonate or an acylamino acid of formula (I) or formula (II) : TO CH3 (CH2 mCONH COOH (I) wherein A is CH2CH2CO2H, CH2CH2SCH3, CH2CO2H, CH2CONH2, CH2CH2CONH2, CH (CH3) 2, CH2CH (CH3) 2, CH (CH3) CH2CH3, CH2C6H5, CH2C6H4OH or CH2OH and m is an integer from 8 to 16; CH3 (CH2) m CONH (CH2) nCO2H (II) wherein n is an integer from 1 to 5 and m is an integer from 8 to 16. In a preferred embodiment, the invention relates to a method for solubilization and naturalization of a recombinant somatotropin using small amounts of biodegradable detergents that are easily removed from the renatured somatotropin by diafiltration, such as C-io- or C-? 2 acylglutamate; N-lauroyl sarcosine, N-decyl sulfate (NDS) of lauroylethylenediamine triacetic acid (LEDA).

DESCRIPTION OF THE PREFERRED MODALITIES The present invention relates to a method for the naturalization of a somatotropin comprising the step of contacting a somatotropin with a detergent composition and water under effective conditions to obtain a renatured somatotropin, wherein the detergent composition contains acylglutamate from Cío to C-? 8, C14, C14 or C18 alkyl sulfate, an alcoholethoxysulfate, lauroylethylenediaminetriacetic acid (LEDA), a linear alkylbenzene sulfonate of C10 to C? S, diphenyldisulfonate or an acylamino acid of formula (I) or (II): (I) wherein A is CH2CH2CO2H, CH2CH2SCH3, CH2CO2H, CH2CONH2, CH2CH2CONH2, CH (CH3) 2, CH2CH (CH3) 2, CH (CH3) CH2CH3) CH2C6H5 > CH2C6H4OH or CH2OH and m is an integer from 8 to 16: CH3 (CH2) m CONH (CH2) n CO2H (II) where n is an integer from 1 to 5 and m is an integer from 8 to 16.

The detergent composition also contains an N-alkylated derivative of formula (I) or (II). The invention further relates to a method for the solubilization and naturalization of a somatotropin containing the steps of the contact of a somatotropin with a detergent composition and water under conditions effective to solubilize the somatotropin., and subsequently adjusting the pH of the resulting somatotropin solution to renaturalize the somatotropin, wherein the detergent composition contains Cg to Cg to acylglutamate, an alkyl sulfate of C, C- | or C, an alkoxylethoxysulfate, lauroylethylenediaminetriacetic acid (LEDA), a linear alkylbenzene sulphonate of C-? 0 to C? s, diphenyldisulfonate or an acylamino acid of formula (I) or (II): (I) wherein A is CH2CH2CO2H, CH2CH2SCH3, CH2CO2H, CH2CONH2, CH2CH2CONH2, CH (CH3) 2, CH2CH (CH3) 2, CH (CH3) CH2CH3) CH2C6H5, CH2C6H4OH or CH2OH and m is an integer from 8 to 16; CH3 (CH2) m CONH (CH2) n CO2H. (II) wherein n is an integer from 1 to 5 and m is an integer from 8 to 16. The detergent composition may also contain an N-alkylated derivative of formula (I) or (II). For the purposes of the present invention, the following terms should be considered with the following definitions. The term "somatotropin" includes mammalian somatotropins, such as bovine, porcine, human, sheep, equine, canine and feline somatotropin, and others such as bird somatotropin. Somatotropins, for the purposes of this disclosure, include somatotropin proteins having naturally occurring sequences, analogs and homologues of the naturally occurring protein that has bioactivity similar to that of somatotropin, ie, they bind to recipients of somatotropin in the animal with the affinity necessary to increase the juvenile growth rate, efficiency in lactation and / or feeding. Somatotropins also include naturally occurring variants of somatotropin that have been elongated, shortened, substituted and / or fused to another protein, as long as said variants are subjected to solubilization and / or naturalization according to the method of the invention. "Recombinant" proteins, also known as heterologous proteins, are proteins that are not normally produced by the host cell. Recombinant DNA technology has allowed the expression of relatively large amounts of heterologous proteins from transformed host cells. For example, the expression of recombinant somatotropins of a variety of animals by transformed microorganisms. Some examples include Goeddel, et al., "Direct Expression of Escherichia coli of a DNA Sequence Coding for Human Growth Hormone," Nature 281: 544-48 (1979) and Seeburg, et al., "Efficient Bacterial Expression of Bovine and Porcine. Growth Hormones, "DNA 2: 37-45 (1983). The production of somatotropins in transformed microorganisms can be achieved by a variety of recombinant genetic plasmids. Examples include those described in UK patent application GB 20732445A and Schoner et al., "Role of mRNA Translational Efficiency in Bovine Growth Hormone Expression in Escherichia coli." PNAS USA 81: 5403-07 (1984). Analogues of bST are also known, for example, as shown in European patent application No. 103,395. The production of bST in transformed microorganisms in addition to E. Coli has been reported by Gray, and others, "Synthesis of Bovine Growth Hormone by Streptomyces lividans." Gene 32: 21-30 (1984), and in the patent E.U.A. No. 4,443,539 for yeast. The "inclusion bodies", also known as "refractile bodies", are the aggregates and cytoplasmic oligomers that contain the recombinant somatotropin to be recovered. A "host cell" is a microbial cell such as bacteria, yeast or other suitable cells such as animal and plant cells that have been transformed to express the recombinant somatotropin. An exemplary host cell is E. Coli K12 (strain W3110 / pBGH-1) which has been transformed to allow the expression of bovine somatotropin. "Naturalization" involves the formation of the correct disulfide bonds so that the somatotropin protein is biologically active at the end of the naturalization step or after further purification. "Doubling" refers to the return of the overall conformational form of the protein sufficient to allow proper oxidation.

"Oxidation" refers to the formation of intramolecular disulfide bonds generally required for a biologically active conformation, preferably the biologically active conformation in its natural form. "Biological activity" means that the somatotropin in question is capable of effecting its desired in vivo physiological response. Biological activity can be determined in the absence of in vivo tests on particular species by performing suitable bioassays. A suitable bioassay for somatotropins is the "rat weight gain bioassay". In this bioassay, the bioactivity of the somatotropin preparations is evaluated according to a known preparation (ie, natural somatotropin extracted) by relating the amount of weight gain of hypophysectomized rats with varying amounts of the preparation administered. The methods for the solubilization and naturalization of somatotropins according to the invention can be applied to any type of recombinant somatotropin, particularly bovine somatotropin. (bST), porcine somatotropin (pST) and human somatotropin (hST). The transformation, cultivation and fermentation of the host cells to produce recombinant somatotropin can be carried out by conventional methods. Recombinant somatotropin, generally in the form of inclusion bodies, can also be recovered from the host cell culture by conventional techniques that fragment the cell in order to release the inclusion bodies, and subsequently the inclusion bodies can be collected as a pelle by differential centrifugation. The detergent composition to be used in the method of the invention generally contains a detergent or combination of detergents that promote the solubilization of somatotropin obtained from the host cell and / or naturalize the somatotropin molecules. Preferably, the detergent used in the methods of the invention is also easily removed from the somatotropin solution at the end of the naturalization step. The detergent composition to be used in the methods of the invention may contain one or more compounds selected from a C 1 to C-is acylglutamate, Cm, Cu, C 1 alkylsulfate, or C 1 alkoxysulfate, lauroylethylenediaminetriacetic acid (LEDA), a linear alkylbenzene sulphonate of C-io a Cis, diphenyldisulfonate or an acylamino acid of formula (I) or (II): (I) wherein A is CH2CH2CO2H, CH2CH2SCH3;. CH2CO2H, CH2CONH2. CH2CH2CONH2, CH (CH3) 2, CH2CH (CH3) 2, CH (CH3) CH2CH3;. CH3C6H5, CH2C6H4OH, or CH2OH and m is an integer from 8 to 16: CH3 (CH2) mCONH (CH2) nCO2H (II) wherein n is an integer from 1 to 5 and m is an integer from 8 to 16. The detergent composition may also contain an N-alkylated derivative of the formula (I) or (II). In a preferred embodiment, the detergent is an acylglutamate of C10 to C? S or a salt, and most preferably an acylglutamate of C10, C-? 2, C-iß or Cie or a salt thereof. For example, the acyl radical can be cocoyl, lauroyl, stearoyl or their respective mixtures. In a preferred embodiment, the acylglutamate is a triethanolamine, sodium or potassium acylglutamate salt. Most preferably, acylglutamate is sodium lauroylglutamate (available as AMISOFT LS-II or AMISOFT LS-II (F) from Ajinomoto), sodium cocoylglutamate (available as AMISOFT CS-II or AMISOFT CS-II (F) from Ajinomoto), TEA cocoylglutamate (available as AMISOFT CT-12 or AMISOFT CT-12S from Ajinomoto), lauroylglutamate from TEA (available as AMISOFT LT-12 from Ajinomoto), hydrogenated sodium seboylglutamate (available as AMISOFT HS-II or AMISOFT HS-II ( F) of Ajinomoto), hydrogenated sodium seboylglutamate with sodium cocoylglutamate (available as AMISOFT GS-II or AMISOFT GS-II (F) from Ajinomoto), hydrogenated disodium seboylglutamate (available as AMISOFT HS-21 from Ajinomoto), potassium cocoylglutamate (available as AMISOFT CK-II or AMISOFT CK-II (F) from Ajinomoto), cocoylglutamic acid (available as AMISOFT CA from Ajinomoto) or hydrogenated seboylglutamic acid (stearoylglutamic acid ) (available as AMISOFT HA from Ajinomoto). Most preferably, the detergent composition contains an acylglutamate of sodium lauroylglutamate, sodium hydrogenated tallowylglutamate, sodium cocoylglutamate, disodium cocoylglutamate or a combination. Most preferably, the detergent composition contains C 1 or C 2 C acylglutamate, and in particular, sodium lauroylglutamate. The detergent may also be an N-alkylated derivative of the formula (I) or (II), such as N-methyl acylglutamate, N-methyl acylaspartate or an N-methyl derivative of Sarkosyl or Amilite. The acylglutamate can be added to the somatotropin mixture, for example, as a powder or as a solution. For example, acylglutamate can be prepared as a 10% solution containing a pH of about 6.9-7.0 by adding 0.84 L H2O 61 ml. 10% NaOH, and 100 g of acylglutamate powder. During the steps of solubilization and naturalization, acylglutamate is preferably present throughout the mixture in a concentration ranging from about 0.2 to about 5% by weight, based on the total naturalization mixture, and most preferably about 0.3 to about 3%. The use of acylglutamates in the detergent composition of the methods of the invention provides a number of economic advantages and environmental, since the acylglutamates are available at low cost and high purity and are easily biodegradable. The use of some acylglutamates is also added to the general economy of the methods of the invention since they are easy to separate from the somatotropin mixture. naturalized, particularly by diafiltration, thus reducing equipment and operating costs. Alternatively, the detergent composition used in the methods of the invention contains a Cι, C 14 or C-is alkyl sulfate, such as NDS; an alcoholetoxysulfate such as C12H25O (CH2CH2O) SO3Na or C? 2H25O (CH2CH2O) 3SO3Na (available from Stepan Co. as Steol ™ CS-130, CS-330 or CS-430) or C14H29 (CH2CH2O) 3SO3Na (available from Henkel Corp. as Standapol ™ ES-40); LEDA (available as Hampshire Corp. as Hampshire LEDA); a linear alkylbenzene sulfonate of C 10 to C 8, such as a linear dodecylbenzenesulfonate; diphenyldisulfonate or an acylamino acid of formula (I) or (II): (I) where A is CH2CH2CO2H, CH2CH2SCH3;. CH2CO2H, CH2CONH2.

CH2CH2CONH2, CH (CH3) 2, CH2CH (CH3) 2, CH (CH3) CH2CH3;. CH2C6H5, CH2C6H4OH, or CH2OH and m is an integer from 8 to 16: CH3 (CH2) mCONH (CH2) nCO2H (II) where n is an integer from 1 to 5 and m is an integer from 8 to 16. The steps of solubilization and naturalization can be performed at the same volume and concentration of detergent. Alternatively, the solubilization step can be performed at a lower volume than the final naturalization step as solubilization of the protein in question, which can be facilitated at higher detergent concentrations. The somatotropin solution can be diluted to obtain the final naturalization volume. For example, the somatotropin can be solubilized in a detergent solution with half the final naturalization volume, then a quantity of water or a pH regulator is added to obtain the volume of complete naturalization. The pH of the solubilization step is preferably from about 9 to about 13, and most preferably ranges from about 10 to about 12. The pH of the naturalization solution preferably ranges from 8 to about 12, most preferably from about 9 to about 11 and still most preferably about 9.5 to about 10.5. The naturalized somatotropin obtained by the methods of the invention is preferably obtained in a yield of at least about 80%, for example, in a yield of about 85% to 95%, wherein the yield is defined as [naturalized somatotropin monomer] / [total reduced somatotropin monomer].

A catalyst, such as cystine or cysteine, can be optionally added to the somatotropin composition of the naturalization step to increase the rate of formation of the disulfide bonds. The detergent composition used for the solubilization and / or naturalization of the somatotropin is preferably removed from the somatotropin solution after the naturalization step to avoid interference with the downstream purification steps. The detergent composition can be removed by a variety of methods, including ion exchange, dialysis or combinations of these techniques. In a preferred embodiment, the detergent is removed from the naturalized somatotropin composition by diafiltration. In a further preferred embodiment, the diafiltration is performed with a cellulose membrane. The removal of detergents such as C 1 and C 2 acylglutamate, N-lauroyl sarcosine, NDS or LEDA by diafiltration is a significant processing advantage since certain detergents are removed from the somatotropin mixture with significantly fewer turnover volumes than the known procedures. When some detergents are removed by simple diafiltration, they require more than 80 volumes of the diafiltration pH regulator. This large volume is a disadvantage in the processing by the size that is required of the diafiltration equipment. In contrast, it has been found that C-io and C12 acylglutamates, N-lauroyl sarcosine, NDS, and LEDA are easy to remove with Rotation volumes less than 30. The acylglutamates of Cío and C? 2 are particularly willing to accept removal by diafiltration and are preferably removed in about 10 to 20 volumes of rotation. The precipitation of impurities and the purification of the naturalized somatotropin composition having the detergent composition substantially removed can be carried out by conventional methods. For example, Storrs et al., Patent of E.U.A. No. 5,182,369 and Bentle et al. Patent of E.U.A. No. 4,652,630, which are incorporated herein by reference, refer to methods for purifying and recovering biologically active somatotropin monomers from solutions that precede the solubilization and naturalization of host cell inclusion bodies produced by a recombinant DNA methodology. The methods of the present invention are part of a broader technique for producing a somatotropin product suitable for parenteral application to target animals. The following examples are included to demonstrate the preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques shown in the following examples represent techniques discovered by the inventors to work well in the practice of the invention, and therefore can be considered to constitute preferred modes for their practice. However, those skilled in the art should, based on the present description, appreciate that many changes can be made in the specific modalities that are shown and still obtain a similar result without having to deviate from the spirit and scope of the invention.

EXAMPLES Four varieties of acylglutramate and lauroylethylenediaminetriacetic acid (LEDA) were tested and showed to be highly efficient to solubilize bovine somatotropin inclusion bodies (bST) and allow the correct naturalization of the bST molecules (examples 1-3). Examples are also provided for the solubilization and naturalization of porcine somatotropin (bST) (examples 3-4).

EXAMPLE 1 Solubilization of bST inclusion bodies An aqueous suspension of bST inclusion bodies was tested and determined to contain 45 g bST / L. The bST used was the N-methionyl derivative of bST which has a native amino acid sequence beginning with phenylalanine. These solubilization experiments were carried out at room temperature, with a pH of the solution of 11.5 and in 20 g bST / L. The solubilization was carried out at half the volume of naturalization, that is, if the naturalization is carried out in a detergent at 1% and in 10 g. bST / L, the solubilization is carried out in a 2% detergent and in 20 g bST / L. The volume was doubled after solubilization to begin naturalization. Water, detergent and NaOH were combined. A sufficient amount of the base was added, so that after the inclusion bodies were added and dissolved, the pH was 11.5, as determined in a preliminary experiment. The mixture of water, detergent and base was stirred as the inclusion bodies were added, and stirred for 30 minutes.

EXAMPLE 2 Naturalization of bST The naturalization step was carried out at room temperature. After the solubilization step, the pH was adjusted to 9.5 by adding HCl, which also served to almost double the volume. Then water was added so that the solution reached its final volume. Cystine was added to reach a final concentration of 1 mM cystine. The mixture was stirred overnight to complete the naturalization process. Examples 1 and 2 were directed to four different detergent compositions acylglutamate, sodium lauroylglutamate, sodium hydrogenated tallowylglutamate with sodium cocoylglutamate, sodium hydrogenated tallowylglutamate and disodium cocoylglutamate, and LEDA as well as a sodium lauroyl sarcosinate comparative detergent. Tables 1 and 2 below summarize the naturalization efficiencies in various pH conditions and detergent concentrations.

TABLE 1 Naturalization Efficiencies (%) for Solubilization pH of 11.5 v Naturalization pH of 9.5 Detergent concentrations Comparative example TABLE 2 Naturalization efficiencies (%) at various Naturalization pH values Comparative example EXAMPLE 3 Solubilization and naturalization of pST PST was solubilized at half the volume of final naturalization in 3% lauroyl acylglutamate, 20 g / l pST, pH 11 to 11.5 at room temperature. The pST used was that which had the amino acid sequence native starting with phenylalanine and having an additional alanine at the amino terminus. After solubilization, 15 to 30 minutes, the pH was adjusted with HCl, diluted with deionized water for the final volume of naturalization, followed by the addition of the cystine catalyst. The final naturalization conditions were 10 g / L pST, 1.5% acylglutamate, 1 mM cystine, pH 9.5, at room temperature. The naturalization was completed in a few hours.

EXAMPLE 4 Removal of the acylglutamate detergent from the renatured pST.

The lauroylacylglutamate detergent used in the naturalization step was removed by diafiltration rather than pH precipitation to remove the impurities. The acylglutamate was suitably removed with 15 to 20 rotational volumes of the cold diafiltration pH regulator of 1 mM NaOH in deionized water. Although the methods of this invention have been described in terms of preferred embodiments, it will be clear to those skilled in the art that variations may be applied to the method described herein without departing from the concept, spirit and scope of the invention. Said clear substitutions and similar modifications for those skilled in the art are considered within the spirit, scope and concept of the invention.

Claims (54)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for the naturalization of a somatotropin comprising: contacting a somatotropin with a detergent composition and water under effective conditions to obtain a naturalized somatotropin, further characterized in that the detergent composition comprises an acylglutamate of Cio, C12, Cie or Cis, a C10 alkyl alkylsulfate, Cu, or Cie, an alkoxylethoxysulfate, lauroylethylenediaminetriacetic acid (LEDA), a linear C-io to C ?8 alkylbenzene sulphonate, diphenyldisulfonate or an acylamino acid of the formula (I) or (II): TO (I) wherein A is CH2CH2CO2H, CH2CH2SCH3, CH2CO2H, CH2CONH2) CH2CH2CONH2, CH (CH3) 2, CH2CH (CH3) 2, CH (CH3) CH2CH3, CH2C6H5, CH2C6H4OH or CH2OH and m is an integer from 8 to 16; or CH3 (CH2) mCONH (CH2) nCO2H (II) where n is an integer from 1 to 5 and m is an integer from 8 to 16.

2. - The method according to claim 1, further characterized in that the somatotropin is bovine, porcine or human somatotropin.

3. The method according to claim 1, further characterized in that the yield of naturalized somatotropin is at least 80%.

4. The method according to claim 1, further characterized in that the detergent composition comprises an acylglutamate of Cio, C? 2, C? 6 or Cis.

5. The method according to claim 4, further characterized in that the acylglutamate is a lauroylglutamate, a cocoylglutamate, a tallowylglutamate or a salt thereof.

6. The method according to claim 5, further characterized in that the acylglutamate is sodium lauroylglutamate, sodium cocoylglutamate, TEA cocoylglutamate, TEA lauorylglutamate, sodium hydrogenated tallowylglutamate, sodium hydrogenated tallowylglutamate with sodium cocoylglutamate, tallowylglutamate hydrogenated disodium, potassium cocoylglutamate, cocoylglutamic acid or hydrogenated tallowylglutamic acid.

7. The method according to claim 6, further characterized in that the acylglutamate is sodium lauroylglutamate, sodium hydrogenated tallowylglutamate, sodium cocoylglutamate, sodium hydrogenated tallow glutamate with sodium cocoylglutamate or disodium cocoylglutamate.

8. The method according to claim 1, further characterized in that the detergent composition comprises a C-io, C14 or C-is alkylsulfate

9. The method according to claim 8, further characterized in that the alkyl sulfate is N-decyl sulfate.

10. The method according to claim 1, further characterized in that the detergent composition comprises an alcoholetoxysulfate.

11. The method according to claim 10, further characterized in that the alcoholetoxysulfate is Ci2H25O (CH2CH2?) SO3Na, C ^^ sOIC ^ C ^ O ^ SOsNa or C14H29? (CH2CH2?) 3SO3Na.

12. The method according to claim 1, further characterized in that the detergent composition comprises lauroylethylenediaminetriacetic acid.

13. The method according to claim 1, further characterized in that the detergent composition comprises a linear alkylbenzene sulfonate of C-? 0 to ds.

14. The method according to claim 13, further characterized in that the detergent composition comprises dodecylbenzenesulfonate.

15. - The method according to claim 1, further characterized in that the detergent composition comprises diphenyldisulfonate.

16. The method according to claim 1, further characterized in that the detergent composition comprises an acylamino acid of the formula (I) wherein A is CH2CH2CO2H, CH2CH2SCH3 or CH2CO2H.

17. The method according to claim 1, further characterized in that the detergent composition comprises an acylamino acid of the formula (I) wherein A is CH2CONH2, CH2CH2CONH2, CH (CH3) 2, CH2CH (CH3) 2, CH (CH3) CH2CH3, CH2C6H5, CH2C6H4OH or CH2OH.

18. The method according to claim 1, further characterized in that the detergent composition comprises an acylamino acid of the formula (II).

19. The method according to claim 1, further characterized in that the detergent composition comprises a C10 or C2 acylglutamate and the method further comprises separating acylglutamate from naturalized somatotropin by diafiltration.

20. The method according to claim 19, further characterized in that the diafiltration uses less than 30 rotational volumes to remove substantially all of the acylglutamate from the somatotropin.

21. The method according to claim 1, further characterized in that the detergent composition comprises lauroylethylenediaminetriacetic acid and the method further comprises the separation of lauroylethylenediaminetriacetic acid from the renatured somatotropin by means of diafiltration.

22. The method according to claim 21, further characterized in that the diafiltration uses less than 30 volumes of rotation to remove substantially all the lauroylethylenediaminetriacetic acid from the somatotropin.

23. The method according to claim 1, further characterized in that the conditions include a pH in the range of about 8 to about 12.

24.- The method according to claim 23, further characterized in that the pH is on the scale of around 9.5 to about 10.5.

25. The method according to claim 1, further characterized in that the conditions include a detergent concentration in the range of about 0.2 to about 5% by weight.

26. A method for the solubilization and naturalization of a somatotropin comprising: contacting the somatotropin with a detergent composition and water under conditions effective to solubilize the somatotropin, and adjusting the pH of the resulting somatotropin solution to naturalize the somatotropin, characterized further because the composition detergent comprises an acylglutamate of C14, C12, C-? 6 or Cis, a C10, C14 or C18 alkyl sulfate, an alcoholethoxysulfate, lauroylethylenediaminetriacetic acid (LEDA), a linear alkylbenzene sulfonate of C10 to Cis, diphenyldisulfonate or an acylamino acid of formula (I) ) or (II): TO OR) wherein A is CH2CH2CO2H, CH2CH2SCH3, CH2CO2H, CH2CONH2, CH2CH2CONH2, CH (CH3) 2, CH2CH (CH3) 2, CH (CH3) CH2CH3, CH2C6H5, CH2C6H4OH, or CH2OH and m is an integer from 8 to 16; CH3 (CH2) mCONH (CH2) nCO2H (II) wherein n is an integer from 1 to 5 and m is an integer from 8 to 16.

27.- The method according to claim 26, further characterized in that the somatotropin is somatotropin. of bovine, porcine or human.

28. The method according to claim 26, further characterized in that the yield of naturalized somatotropin is at least 80%.

29. The method according to claim 26, further characterized in that the detergent composition comprises an acylglutamate of C10, C? 2, Cie or C18.

30. The method according to claim 29, further characterized in that the acylglutamate is a lauroylglutamate, a cocoylglutamate, a tallowylglutamate or a salt thereof.

31. The method according to claim 30, further characterized in that acylglutamate is sodium lauroylglutamate, sodium cocoylglutamate, TEA cocoylglutamate, TEA lauroylglutamate sodium hydrogenated tallowylglutamate, sodium hydrogenated tallowylglutamate with sodium cocoylglutamate, disodium tallowglutamate. hydrogenated, potassium cocoylglutamate, cocoylglutamic acid or hydrogenated tallowylglutamic acid.

32. The method according to claim 31, further characterized in that the acylglutamate is sodium lauroylglutamate, sodium hydrogenated tallowylglutamate, sodium cocoylglutamate, sodium hydrogenated tallowylglutamate with sodium cocoylglutamate or disodium cocoylglutamate.

33. The method according to claim 26, further characterized in that the detergent composition comprises a C 1, C or C 8 alkylsulfate.

34.- The method according to claim 33, further characterized in that the alkyl sulfate is N-decyl sulfate.

35. The method according to claim 26, further characterized in that the detergent composition comprises an alcoholetoxysulfate.

36. - The method according to claim 35, further characterized in that the alcoholetoxysulfate is Ci2H25O (CH2CH2O) SO3Na, C2H25 (CH2CH2O) 3SO3Na or Ci 4 H 29 O (CH 2 CH 2 O) 3SO 3 Na.

37. The method according to claim 26, further characterized in that the detergent composition comprises lauroylethylenediaminetriacetic acid.

38. The method according to claim 26, further characterized in that the detergent composition comprises a linear C 1 to Cis alkyl benzene sulfonate.

39.- The method according to claim 38, further characterized in that the detergent comprises dodecylbenzenesulfate.

40. The method according to claim 26, further characterized in that the detergent composition comprises diphenyldisulfonate.

41. The method according to claim 26, further characterized in that the detergent composition comprises an acylamino acid of the formula (I) wherein A is CH2-CH2CO2H, CH2CH2SCH3 or CH2CO2H.

42. The method according to claim 26, further characterized in that the detergent composition comprises an acylamino acid of the formula (I) wherein A is CH2CONH2, CH2CH2CONH2, CH (CH3) 2, CH2CH (CH3) 2, CH (CH3) ) CH2CH3, CH2C6H5, CH2C6H4OH or CH2OH.

43. - The method according to claim 26, further characterized in that the detergent composition comprises an acyl amino acid of the formula (II).

44. The method according to claim 26, further characterized in that the detergent composition comprises an acylglutamate of C-? 0 or C-? 2 and the method further comprises separating acylglutamate from the naturalized somatotropin by means of diafiltration.

45. The method according to claim 44, further characterized in that the diafiltration uses less than 30 rotational volumes to remove substantially all of the acylglutamate from the somatotropin.

46. The method according to claim 26, further characterized in that the detergent composition comprises a lauroylethylenediaminetriacetic acid and the method further comprises the separation of lauroylethylenediaminetriacetic acid from naturalized somatotropin by means of diafiltration.

47. The method according to claim 46, further characterized in that the diafiltration uses less than 30 volumes of rotation to remove substantially all the lauroylethylenediaminetriacetic acid from the somatotropin.

48. The method according to claim 26, further characterized in that the conditions include a pH in the range of about 9 to about 13.

49. - The method according to claim 26, further characterized in that the conditions include a pH in the range of about 8 to about 12.

50.- The method according to claim 49, further characterized in that the pH is in the scale from around 9.5 to about 10.5.

51. The method according to claim 26, further characterized in that the conditions include a detergent concentration in the range from about 0.2 to about 5% by weight.

52. A method for obtaining bioactive somatotropin comprising: contacting a somatotropin with a detergent composition and water under effective conditions to solubilize the somatotropin, adjust the pH of the somatotropin solution to naturalize the somatotropin, and remove the detergent composition from naturalized somatotropin by means of diafiltration, further characterized in that the detergent composition comprises a C 1 or C 2 acylglutamate, N-lauroyl sarcosine, N-decyl sulfate or lauroylethylenediamine triacetic acid.

53. The method according to claim 52, further characterized in that the diafiltration step uses less than 30 volumes of rotation to remove substantially all of the detergent from the somatotropin.

54. The method according to claim 53, further characterized in that the detergent comprises an acylglutamate of C? 2 and the diafiltration step uses 10 to 20 volumes of rotation to remove substantially all the detergent from the somatotropin.