WO2015051431A1

WO2015051431A1 - Method for producing active recombinant bovine somatrotopin, and thus obtained product

Info

Publication number: WO2015051431A1
Application number: PCT/BR2014/000359
Authority: WO
Inventors: Dolivar Coraucci Neto; Flávia BARBOZA CAMARGO
Original assignee: Ouro Fino Saúde Animal Ltda
Priority date: 2013-10-10
Filing date: 2014-10-01
Publication date: 2015-04-16
Also published as: BR102013026129A2

Abstract

The present invention relates to a method for producing somatotropin from inclusion bodies without using denaturing chemicals. More specifically, the present invention relates to a method for producing active bovine somatotropin from recombinant cells that express bovine somatrotopin in high concentrations in inclusion bodies, in that the inclusion bodies are solubilised by means of successive centrifugation steps in water, combined with the use of the tris buffer with an alkaline pH. The present invention also relates to the thus obtained active recombinant bovine somatotropin.

Description

"PROCESS FOR THE PRODUCTION OF ATOTROPI SOUND IN THE ACTIVE RECOMBINANT BOVINE AND OBTAINED PRODUCT"

Field of the Invention

The present invention relates to a process for obtaining somatotropin from inclusion bodies without the use of denaturing chemical agents. More specifically, the present invention relates to a method of obtaining bovine somatotrophin active from recombinant cells expressing high concentrations of bovine somatotropin in inclusion bodies, wherein said inclusion corpuscles are solubilized using successive centrifugations in water combined with the use of Tris buffer at alkaline pH. The active recombinant bovine somatotropin obtained is also the subject of the present invention.

History of the Invention

Heterologous proteins expressed in transformed host cells occur in soluble form, or in the form of inactive and insoluble inclusion bodies (or refractory corpuscles), depending on such factors as the redox environment of the host cell, the level of expression and the nature of the protein. There is also a borderline case where both the soluble and the insoluble forms of the proteins are expressed simultaneously.

Inclusion corpuscles (or refractile corpuscles) are insoluble aggregates of dense, electron-refractory proteins found in the cytoplasm and periplasmic space of recombinant cells expressing high concentrations of heterologous proteins. The inclusion corpuscles are devoid of biological activity and must be submitted to processes of isolation, solubilization, renaturation and purification to become biologically active.

The recovery of bioactive proteins from the inclusion corpuscles involves basically three known steps: isolation of the inclusion corpuscles, solubilization of the protein aggregates and renaturation of the solubilized proteins. The isolation of the corpuscles occurs by mechanical disruption of the cells, followed by centrifugation. Protein aggregates generated are solubilized at high concentrations of chaotropic agents such as urea, guanidine hydrochloride (Gdn-HCl), thiocyanate salts, or detergents such as sodium dodecyl sulfate (SDS), N-cetyl trimethyl ammonium chloride and N-lauryl sarcosine (NLS ). Additional reducing agents such as β-mercaptoethanol, dithiotreitol or cysteine are commonly added to aid in the solubilization of inclusion bodies, maintaining the cysteine residues in the reduced state, preventing the formation of undesirable intra and intermolecular disulfide bonds. Chelating agents such as ethylenediamine tetraacetic acid (EDTA) may also be added to the solubilization buffer to prevent oxidation of the cysteine residues. Solubilized proteins are then renatured in their natural conformation by removal of the reducing agents in the presence of additives that enhance the production of bioactive proteins such as L-arginine, dimethyl sulfoxide (DMSO) and polyethylene glycol (PEG).

Although bioactive protein recovery methods are widely known in the literature, with minor variations in the type of denaturing agent and the additional reducing agent employed, more lenient methods of solubilizing proteins from inclusion bodies are also known. These milder methods are employed in order to reduce the formation of aggregates generated during the protein renaturation process.

Protein aggregates are formed from undesirable intermolecular interactions between the protein intermediate generated during the first steps of renaturation. These intermediates have exposed hydrophobic bonds capable of binding the denatured proteins formed in the presence of high concentrations of chaotropic agents. One of the ways to reduce protein aggregation is to prevent the formation of hydrophobic interactions during the early stages of renaturation and to increase the renaturation of the proteins in the inclusion corpuscles. This can be achieved if the proteins are solubilized while the natural secondary structures are still intact in the inclusion bodies. In order to maintain the natural secondary structures, thus avoiding the formation of aggregates, processes in which inclusion bodies are solubilized in non-denaturing solutions such as 100 mM Tris / 2 M urea at alkaline pH are known in the literature (Panda et al. , Triton X-100 / DMSO (Jevsevar et al., 2000), Tris 100mM / 6M n-propanol buffer / 2M urea (Panda et al., 2012), 0.2% N-lauryl sarcosine al .; 2005).

Brazilian patent application PI8307086 to Genentech Inc. describes a process for treating a heterologous expression product of a host cell culture by contacting the inclusion corpuscle with denaturing solution.

The Brazilian patent application PI9007992, Pitman-Moore Inc., describes a process for the recovery of recombinant somatotropin by addition of alkaline earth metal salts, separation of the precipitate from the solution and recovery of the recombinant protein from the solution.

International patent application W098 / 29433, to Monsanto Company, discloses a method of solubilizing and / or naturating somatotropin which comprises contacting the latter with a detergent composition and water under conditions for obtaining a naturally occurring somatotropin, wherein the detergent composition may comprises an acyl glutamate Cio, C12, C ₆ or Cia, an alkyl sulfate , C10, C14 or Cie, an ethoxy sulfate alcohol, an ethylene diamine triacetic acid (LEDA) lauroyl acid, linear alkyl benzene sulfonate with C18 -C10 a disulfide diphenyl or an acyl amino acid.

Further, LG Chemical Pat. PI9913018 by LG Chemical discloses a process for the preparation of somatotropin in the absence of chaotropic agents from the solubilization of inclusion bodies with alcohol solution at alkaline pH followed by contact of the solubilized protein with weak oxidizing agent 2-mercaptoethanol.

In addition to the time spent with renaturation processes, it is estimated that the total yield of bioactive proteins obtained from corpuscles of inclusion using the traditional methods mentioned above is low, around 15 - 25%, which impacts greatly on their production costs.

The solubilization process of the present invention is novel in that it uses successive washes of inclusion corpuscles followed by solubilization of said corpuscles with non-aggressive denaturing agents allowing the maintenance of the natural secondary structure of the retained protein in the inclusion corpuscles. Accordingly, the denaturation / renaturation steps of the protein are eliminated as well as the use of additional reducing agents, making the process of the present invention faster and more economically advantageous than the processes known in the art.

Brief Description of the Invention

The present invention provides the obtainment of recombinant bovine somatotropin active from inclusion bodies using successive centrifugations of the inclusion bodies in water followed by solubilization in Tris buffer pH alkaline capable of extracting the protein of interest in the active form rapidly without the denaturation / renaturation steps and without the use of chaotropic denaturing agents, as follows:

(a) washing of the inclusion bodies in water and separation of the insoluble fractions by successive centrifugations;

(b) solubilization of inclusion corpuscles in non-denaturing solution of Tris buffer at alkaline pH.

Description of Figures

Figure 1 illustrates the determination of the secondary structure by circular dichroism. In dashed line: rbGH standard; in continuous line: rbGH OF. We can observe a secondary structure with 60 - 70% similarity to standard.

Figure 2 shows the SDS-PAGE image of the cell disruption cycles by HAP. In the image we can observe the following samples: a. 4 μΙ standard molecular weight marker (GE LMW); B. 5 μΙ of standard rbGH (0.4 pg / pL); W. 2 pl of solubilized in Tris; d. 2 pl of solubilized 2M Urea; and. 2 pl of solubilized 4M Urea; f. 2 pl of solubilized 6M Urea; g. 2 pl of solubilized 8M Urea.

Figure 3 illustrates the SDS-PAGE of the cell disruption cycles by HAP. In the image we can observe the following samples: a. 4 pl of standard molecular weight marker (GE LMW); B. 5 pl of rbGH standard (0.4 pg / pL); W. 1 pl of inclusion body centrifuged after PAH; d. 10 μl of the PAH supernatant; and. 10 pl of the first inclusion body wash; f. 10 pl of the second wash of the inclusion body; and g. 1 pl of inclusion body solubilized in Tris-Base.

Detailed Description of the Invention

The inclusion bodies may be produced by processes well known in the literature, such as described in the Brazilian patent PI8307086.

In the proposed new process, after fermentation, the transformed cells are disrupted using a high pressure homogenizer in a 0.05 Tris-disruption buffer at 0.2M, pH 5.0 to 7.5 and 0.01M EDTA, at a concentration of 1 to 10 g / mL, generating a suspension of soluble cell debris and insoluble proteins composed of the inclusion corpuscles.

After cell disruption, 3 to 4 successive washes of the debris are performed with reverse osmosis water followed by 3 to 4 centrifugations at 6000 rpm for 20 to 40 minutes for removal of the water soluble proteins and separation of the inclusion corpuscles. The inclusion corpuscles are solubilized in 0.1M Tris buffer pH 0.5 to 12.5 at a final concentration of 5 to 50 mg / mL under stirring at room temperature for two hours. After solubilization, the active recombinant bovine somatotropin is centrifuged one last time at 6000 rpm for one hour to remove the undesirable insoluble solids remaining after solubilization. Upon completion of the solubilization, somatotropin can be purified by standard chromatographic methods. The process according to the present invention does not use chaotropic denaturants, so it maintains the secondary structure (Figure 1) of the proteins intact and no further steps of renaturation are required for recovery of protein function, which ultimately results in a faster process .

In addition, the process of the present invention is more economical, since 20 ml of alkaline pH Tris solubilisation buffer is required for each gram of inclusion corpuscle used, whereas in the traditional process 20 ml of urea denaturing agent is required, however. to 80 times more concentrated (2 to 8 M) per gram of inclusion corpuscle. The yield data point to a final yield after purification of 70-75% bioactive protein.

Examples of tests performed for confirmation of the novel features of the recombinant active bovine somatotropin and process obtained according to the present invention are presented below.

Example 1: Evaluation of HAP break cycles

The PAH disruption process involves 3 cycles of 10 minutes each, where in cycle 1 the temperature ranges from 12 to 26 ° C at a pressure of 1050 bar; in cycle 2 the temperature varies from 12 to 22 ° C at 1000 bar and in cycle 3 the temperature varies from 15 to 24 ° C at 1050 bar. Table 1 below illustrates the results of HAP break cycles.

Table 1: Results of PAH rupture cycles before and after.

HAP

Methods

before after

Disruption

Cell Phone - Yes

rbGH inside rbGH outside

Cell SDS-Page

Dry Weight 17 g / L 3.5 g / L

Size of

particle (DLS) 1212 nm 193 nm

Bradford 0.41 mg / mL 6.9 mg / mL According to Table 1, it is possible to state that the high pressure homogenizer (HAP) is efficient in cell disruption and release of inclusion corpuscles (containing rbGH). Inclusion corpuscles are fully released from inside the cell after the 3 cycles of tear.

Example 2: Evaluation of inclusion solubility of corpuscles

The objective of the study was to determine the best solubilization condition of the inclusion corpuscle of rbGH by fermentation, without losses in protein recovery. For the execution of this protocol was carried out on bench scale fermentation with a working volume of 5 liters. The sample used was inclusion corpuscle containing rbGH. The methods used were: (i) Tris solubilization, where for each gram of wet inclusion corpuscle 20 mL of 100 mM Tris-Base solubilization buffer pH 12.0 was used; (ii) Solubilization with 2 M urea, where 20 mL of 2 M urea solubilization buffer were used for each moist inclusion corpuscle; (iii) Solubilization with 4M urea, where 20 mL of 4M urea solubilization buffer was used for each moist inclusion corpuscle; (iv) Solubilization with 6M urea, where for each gram of wet inclusion corpuscle 20 mL of 6M urea solubilization buffer was used; (v) Solubilization with 8M urea, where 20 mL of 8M urea solubilization buffer was used for each moist inclusion corpuscle.

The analysis methodology included the analysis of the electrophoretic profile:; and quantification of total proteins by Bradford.

All samples were solubilized by shaking with the aid of a magnetic stirrer, and then held at room temperature for 2 hours for complete solubilization. After this period, it was centrifuged at 12,000 rpm, 3 minutes at 4 ° C for clarification of the solubilized sample. Fractions from these samples were separated from each condition for SDS-PAGE analysis (Figure 2) and quantification of total proteins by Bradford.

The results are shown in Table 2 below: Table 2. Summary of the results of the comparison with the cited methods.

According to Table 2 above, it can be concluded that the most efficient method for the solubilization of inclusion corpuscles (containing rbGH) is 0.1 M Tris buffer pH 12.0.

Example 3: Evaluation of washing steps of inclusion corpuscles

The present test addresses the description and evaluation of the rGGH inclusion corpuscle washing steps obtained by the fermentative route in order to determine whether inclusion body washes are resulting in loss of rbGH. Various wash buffers are described in the literature, and these include detergents / surfactants such as Triton X-100, Tween 20% and also, in some cases, salts such as NaCl and KCl, as well as metal chelator such as EDTA.

The use of these compounds is efficient in some conditions and unnecessary in others. In the case of the present invention one opted for simple washing containing only water, which entails a reduction of expenses.

The inclusion corpuscle washing methodology comprises for each gram of wet inclusion body the use of 10 mL of water for washing the inclusion corpuscles.

After homogenization on an orbital shaker, centrifugation was performed in a Tubular Type Centrifuge. This process was repeated twice.

The analysis methodology followed the analysis of the electrophoretic profile and the quantification of total proteins by Bradford. Fractions of these samples were separated from each condition for SDS-PAGE analysis (Figure 3). The results are shown in Table 3 below:

Table 3. Summary of results compared to methods

According to Table 3 presented above, it can be concluded that the method of washing inclusion corpuscles with water in a Tubular Type centrifuge proved to be efficient with respect to the recovery of rbGH in the inclusion body.

Example 4. Evaluation on the performance of two purification processes (precipitation x lyophilization)

The present test deals with the description and evaluation of precipitation and lyophilization to obtain recombinant bovine somatotropin in the form of powder. The objective was to determine the best condition for obtaining recombinant bovine somatotropin, without loss of yield and protein structure. The sample used was 200 mL of solubilized sample of rbGH at a concentration of 30 g / L and 100 mL for each test.

The methodology used included organic solvent precipitation where 100 mL of the sample to be precipitated was reduced to pH 6.0 with the addition of acetic acid under continuous stirring on a bench magnetic stirrer. Acetone was slowly added to that solution until the concentration of 75% (for each part of solution 3 parts of acetone) was reached, kept for 1 hour in a refrigerated environment at 4Â ° C, and then centrifuged at 6,000 rpm for 10 minutes at 4 ° C. The supernatant was discarded and the precipitate was reserved. 200 mL of 95% acetone was added to the precipitate for the wash step, and then centrifuged at 6,000 rpm for 10 minutes at 4 ° C, and the supernatant was discarded again and the precipitate. To the precipitate was added 50 mL of ethyl ether, and then centrifuged at 6,000 rpm for 10 minutes at 4 ° C, and finally the resulting precipitate was dried under refrigeration at 4 ° C. A second aspect of the methodology was the use of the lyophilization process.

The analysis methodology included: i) an analysis of the electrophoretic profile; ii) analysis of total proteins by Bradford quantification of proteins by Bradford; iii) determination of secondary structure by Circular Dyroism; and IV) determination of monomeric constituents by Gel filtration Sephacryl S100.

The volume used for each of the tests was 100 mL, as the protein concentration in that sample was 30 mg / mL, we should find an approximate value of 3 g dry weight. All samples were maintained under the same conditions of temperature and luminosity after the precipitation and lyophilization processes.

The results are shown in Table 4 below:

Table 4. Summary of results compared to methods.

From the results obtained, as shown in Table 4 above, it can be concluded that both methods are efficient for obtaining the recombinant bovine somatotropin active in the form of powder. The drying processes do not interfere with the characterization of the protein at the end and it can be observed:

a) similar content observed by Bradford;

b) presence of the protein on SDS-PAGE; same percentage of monomeric constituents per FPLC; and the same secondary structuring (alpha-helices) observed circular dichroism.

Claims

A process for the production of active recombinant bovine somatotropin from inclusion corpuscles of a recombinant host cell comprising the steps of:

(b) solubilization of inclusion corpuscles in non-denaturing solution of Tris buffer at alkaline pH;

the process being carried out without the use of chaotropic denaturing agents.

A process according to claim 1, characterized in that the transformed cells are disrupted using a high pressure homogenizer in a 0.05 to 0.2 M Tris disruption buffer, pH 5.0 to 7, 5 and 0.01 M EDTA.

A process according to claim 2, characterized in that the tear-off buffer is present at a concentration of 1 to 10 g / ml.

A process according to any one of claims 1 to 3, characterized in that 3 to 4 successive washes of the debris are performed with reverse osmosis water, followed by 3 to 4 centrifugations at 6000 rpm, from 20 to 40 minutes for removal of soluble proteins in water and separation of the inclusion corpuscles.

A process according to claim 1, characterized in that the inclusion bodies are solubilized in 0.01 Tris buffer at 1.0 M, pH 9.0 to 13.0.

A process according to claim 5, characterized in that the inclusion bodies are solubilized in 0.1 to 0.5 M Tris buffer pH 11.0 to 12.5 with stirring at room temperature for at least two hours .

A process according to any one of claims 1, 5 or 6, characterized in that 0.1 to 0.5 M Tris buffer, pH 11.0 to 12.5, has a final concentration of 5 to 50 mg / ml .

A process according to any one of claims 1 to 7, characterized in that after the solubilization the active recombinant bovine somatotropin is centrifuged one last time at 6000 rpm for one hour to remove the undesirable insoluble solids.

A process according to claim 1, characterized in that it eliminates additional steps of renaturation for recovery of protein function.

10. ACTIVE RECOMBINATE BOVINE SOMATOTROPIN, characterized in that it is produced according to the process defined in claims 1 to 9.