Method For Producing A Recombinant Protein Of Interest

Abstract

Disclosed is a method for producing a recombinant protein of interest, characterised in by the following steps: (a) providing a fusion protein comprising an N.sup.pro autoprotease moiety and a protein of interest moiety in inclusion bodies, (b) solubilising the fusion protein in the inclusion bodies by subjecting the inclusion bodies to a solubilisation buffer containing a detergent and wherein the solubilisation buffer contains no chaotropes or chaotropes in a concentration of less than 1.5 M urea (c) refolding the solubilised fusion protein and (d) allowing the fusion protein to be cleaved by the N.sup.pro autoprotease moiety under kosmotropic conditions, wherein the recombinant protein of interest is cleaved from the fusion protein, and (e) recovering the protein of interest.

Claims

1. Method for producing a recombinant protein of interest, characterised in by the following steps: (a) providing a fusion protein comprising an N.sup.pro autoprotease moiety and a protein of interest moiety in inclusion bodies, (b) solubilising the fusion protein in the inclusion bodies by subjecting the inclusion bodies to a solubilisation buffer containing a detergent and wherein the solubilisation buffer contains no chaotropes or chaotropes in a concentration of less than 1.5 M urea (c) refolding the solubilised fusion protein and (d) allowing the fusion protein to be cleaved by the N.sup.pro autoprotease moiety under kosmotropic conditions, wherein the recombinant protein of interest is cleaved from the fusion protein, and (e) recovering the protein of interest.

2. Method according to claim 1, characterized in that the inclusion bodies were generated in a recombinant production system.

3. Method according to claim 1, characterized in that the detergent is contained in the solubilisation buffer in a concentration of 0.2 to 15% (w/v).

4. Method according to claim 1, characterized in that steps (c) and/or (d) are performed at kosmotropic conditions that correspond to a urea concentration of 0 to 1.5 M.

5. Method according to claim 1, characterized in that steps (b), (c) and/or (d) are performed in a buffer.

6. Method according to claim 1, characterized in that steps (b), (c) and/or (d) are performed in a buffer containing a reducing agent.

7. Method according to claim 1, characterized in that steps (c) and/or (d) are performed in a buffer containing an ion chelating agent.

8. Method according to claim 1, characterized in that the detergent in the solubilisation buffer is a non-ionic detergent, or an anionic detergent, or mixtures thereof.

9. Method according to claim 1, characterized in that steps (c) and/or (d) are performed in a buffer containing a detergent; and/or an amino acid; and/or a carbohydrate; or mixtures thereof.

10. Method according to claim 1, characterized in that steps (c) and/or (d) are performed in a buffer with a pH of 6 to 9.5.

11. Method according to claim 1, characterized in that the protein of interest is a protein for therapeutic use in humans.

12. Method according to claim 1, characterized in that steps (c) and/or (d) are performed in the presence of a buffer comprising NaCl.

13. Method according to claim 1, characterized in that that steps (c) and/or (d) are performed in the presence of a buffer comprising sucrose, DTT, NaCl, EDTA and a detergent.

Description

[0067] The present invention is further described by the following examples and the drawing figures, yet without being restricted thereto.

[0068] FIG. 1: Renaturation of fusion protein 1 with and without chaotropes.

[0069] FIG. 2: Renaturation of fusion protein 1 in dependence of the NLG concentration in the renaturation batch.

[0070] FIG. 3: Kinetic of renaturation of fusion protein 1 with and without chaotropes.

[0071] FIG. 4: Renaturation of fusion protein 1 in dependence of the NLS concentration and L-Arginine in the renaturation batch.

[0072] FIG. 5: Renaturation of fusion protein 1 in dependence of the residual NLS concentration and the used renaturation buffer.

[0073] FIG. 6: Renaturation of fusion protein 2 in dependence of the NLS, urea and fusion protein in the renaturation batch.

EXAMPLES

Materials and Methods

Results

[0074] This summary describes the comparison of the renaturation of two different N.sup.pro fusion proteins in chaotrope and detergent containing buffer.

[0075] The solubilization of N.sup.pro fusion protein containing inclusion bodies in chaotrope (e.g. urea and guanidine hydrochloride) containing buffers and the sequential renaturation by rapid dilution is a common process in the production of recombinant therapeutic proteins. Here we show the solubilization and renaturation of two different N.sup.pro fusion proteins in detergent (e.g. N-Lauroyl sarcosinate and N-Lauroyl-glutamate) containing buffers.

Materials and Methods

[0076] All used chemicals were of Ph. Eur. Grade and were purchased by known suppliers.

[0077] The inclusion bodies from high cell density cultures of E. coli were extracted from the bacteria by French press and continuous centrifugation. The Inclusion bodies were washed with purified water. One part of the pre-diluted inclusion were mixed with two parts of the respective solubilization buffer and stirred at room temperature for 30 minutes. Afterwards one part of the solubilized inclusion bodies was mixed with four parts of renaturation buffer. The renaturation batch for fusion protein 1 was further stirred at 2-8 C. and the renaturation batch of fusion protein 2 was stirred at room temperature.

Chemicals:

[0078] All used substances were Ph. Eur. grade or of comparable quality. The buffers were prepared with purified and de-ionized water.

Substance: Supplier

1, 4-Dithiotreitol (DTT): C.F.M. Tropitzsch

[0079] 2-Amino-2-(hydroxymethyl) propane-1,3-diol (Tris): Angus Chemie
3-(N-Morpholino)-Propanesulfonacid-sodium salt: Sigma-Aldrich

3-(N, N-Dimethylpalmitylammonio)propanesulfonat (Zwittergent 3-14): Sigma-Aldrich

Acetic Acid (80%): Merck

[0080] Ethylendiaminetetraacedic acid, 2 Na (EDTA): Merck
Guanidine hydrochloride: Sigma-Aldrich
Hydrochloride acid (HCL): Merck
L-Arginine hydrochloride: Ajinomoto
Polyethylene glycol hexadecyl ether (Brij 58): Sigma-Aldrich

Polysorbate 80: BioRad

[0081] N-lauroyl glutame: Ajinomoto
Sodium N-lauroyl sarcosinate: Sigma-Aldrich
Sodium chloride (NaCl): Merck/Baker

Sucrose: Suedzucker AG

Urea: Merck

Analytical Methods:

[0082] The analytical determination of the content of fusion protein, cleaved N.sup.pro and cleaved model protein was performed on a HPLC system with Autosampler and multiple wavelength detector (Agilent Technologies, Santa Clara, USA). The determination of contents of model protein 1 was performed with a Zorbax 300SB-C3 (Agilent Technologies, Santa Clara, USA) column with a bed height of 15 cm and a diameter of 4.6 mm. The particle diameter was 3.5 m and the pore size 300 . The samples were diluted with sample dilution buffer (100 mM MOPS, 7 M guanidine hydrochloride, 2% (w/v) Zwittergent 3-14, 130 mM DTT, pH 7.0) to a target concentration of 150 g/mL. The measurements were performed with a constant linear flow of 1.5 mL/min at 60 C. The determination of contents of model protein 2 was performed with a Tosoh TSK Super Octyl (Tosoh Biosciences, Tokyo, Japan) column with a bed height of 10 cm and a diameter of 4.6 mm. The particle diameter was 2 m. The samples were diluted with sample dilution buffer (50 mM Tris, 7 M guanidine hydrochloride, 0.5% (w/v) Polysorbate 80, 100 mM DTT, pH 8.0) to a target concentration of 225 g/mL. The measurements were performed with a constant linear flow of 1.1 mL/min at 50 C. The determination of the amount of released/cleaved off fusion partner was determined by ion paired reversed phase high performance chromatography. A five point calibration curve (peak area vs. measured concentration) was performed with the purified fusions partner. The cleavage yield was determined by the following equation:

[00001]$Y=\frac{c_{\mathrm{fp},24}.Math.h}{c_{\mathrm{FP}}.Math.x_{\mathrm{fp}}}.Math.100,$

with
C.sub.fp,24h Concentration of the fusion partner after 24 h refolding [mg/mL]
C.sub.FP Concentration of the fusion protein at refolding start [mg/mL]
x.sub.fp Mass fraction of the fusion partner in the fusion protein, calculated according to

[00002]$x_{\mathrm{fp}}=\frac{{\mathrm{MW}}_{\mathrm{FP}}-{\mathrm{MW}}_{N^{\mathrm{Pro}}}}{{\mathrm{MW}}_{\mathrm{FP}}},$

with
MWFP Molecular weight of the fusion protein [Da]
MW.sub.N.sup.pro Molecular weight of N.sup.pro moiety of the fusion protein [Da]
The buffers used are listed in Table 1.

TABLE-US-00001 TABLE 1 Buffers used for solubilization and renaturation of the used N.sup.pro fusion proteins Buffer Composition Solubilization 1 75 mM Tris, 4.5M urea, 37.5 mM DTT, pH 7.9 Solubilization 2 75 mM sodium phosphate, 3% (w/v) N-lauroyl glutamate, 37.5 mM DTT, pH 7.9 Solubilization 3 75 mM Tris, 4.5M urea, 3% (w/v) N-lauroyl glutamate, 37.5 mM DTT, pH 7.9 Renaturation 1 0.625M L-Arginine, 50 mM Tris, 25 mM DTT, 1.25M NaCl, (reference) 6.25 mM EDTA, 0.00625% (w/v) Brij58, 0.625M Sucrose, pH 8.0 Renaturation 2 600 mM Urea, 0.625M L-Arginine, 50 mM Tris, 25 mM DTT, (+urea) 1.25M NaCl, 6.25 mM EDTA, 0.00625% (w/v) Brij58, 0.625M Sucrose, pH 8.0 Renaturation 3 0.4% (w/v) N-lauroyl glutamate, 0.625M L-Arginine, 50 mM (+NLG) Tris, 25 mM DTT, 1.25M NaCl, 6.25 mM EDTA, 0.00625% (w/v) Brij58, 0.625M Sucrose, pH 8.0 Renaturation 4 50 mM Tris, 630 mM NaCl, 940 mM sucrose, 0.012% (w/v) Brij 58, 20 mM DTT, pH 8.0; Renaturation 5 50 mM Tris, 20 mM DTT, 1.25M NaCl, 6.25 mM Na-EDTA, 0.00625% (w/v) Brij 58, 625 mM sucrose, pH 8.0

Results:

Fusion Protein 1

[0083] The fusion protein used in this example (model protein 1) consists of 338 amino acids. The 6-His-NPro-EDDIE-q15 moiety is 161 amino acids and the fusion partner consists of 177 amino acids. A 6-His tag was fused to the N-terminus of the N.sup.pro to enable the purification with a metal chelate affinity chromatography. Based on the amino acid sequence the isoelectric points are determined with 5.92 and 4.99 for the fusion protein and the fusion partner. The molecular masses are calculated with 18350.6 Da for 6-His-NPro-EDDIE-q15, 18893.2 Da for the fusion partner and 37243.8 Da for the fusion protein. The fusion partner has three cysteines (Cys71, Cys89 and Cys122). Cys71 and Cys89 form one intramolecular disulfide bridge. A false bridging with the Cys 122 was not shown.

[0084] The renaturation yield in the buffer even containing urea is the reference for the other determined solubilization and renaturation approaches. With additional urea in the renaturation buffer the cleavage yield decreases up to 15%. The higher chaotrope concentration inhibits the folding reaction. Additional NLG in the renaturation buffer lifts the renaturation yield up to 15%. The renaturation after solubilization of the inclusion bodies in NLG containing buffer achieved in comparison to the urea based renaturation of fusion protein 1 up to relatively 65% higher yields. The ability of NLG to stabilize the partial and unfolded fusion protein monomers is significant higher compared to urea. The positive effect of NLG during refolding was slightly decreased through the addition of additional urea and NLG in the renaturation buffer. The combined solubilization with urea and NLG resulted in lower yields than the NLG based renaturation.

[0085] FIG. 2 shows the results of the solubilization of the inclusion bodies and the renaturation of fusion protein 1 in renaturation buffer 1 with different residual concentrations of NLG.

[0086] The solubilization capability of NLG is fully given above the critical micelle concentration of the detergent. With increasing residual NLG concentration in the renaturation batch the refolding yield decreases. For the renaturation of fusion protein 1 an optimum for the residual NLG concentration was determined between 0.3 and 0.4% (w/v). In this concentration range the full ability of solubilizing of the inclusion bodies is given.

[0087] FIG. 3 shows the comparison of the cleavage kinetics of the chaotrope and detergent based renaturation of fusion protein 1 in renaturation buffer 1 at a fusions protein concentration of 4 mg/mL. The solubilization was done in solubilization buffer 1 and solubilization buffer 2.

[0088] The renaturation kinetic proceeds equal in the first 8 hours of the renaturation process. Afterwards the kinetics diverges. The renaturation reaction in the urea based renaturation was finished but the refolding in the NLG containing batch continued. At the end of the monitored time range (24 h) a yield of 72% and 94% was reached for the urea and NLG based renaturation of fusion protein 1.

[0089] A second examined detergent for the chaotrope free renaturation of N.sup.pro fusion proteins was NLS. The results for the renaturation in dependence of the residual NLS concentration and L-Arginine in the renaturation buffer are shown in FIG. 4. The solubilization of the inclusion bodies was performed with an adapted solubilization buffer 1, NLG was exchanged against NLS. The renaturation was performed with renaturation buffer 1, with and without the given concentration of L-Arginine

[0090] The renaturation yields are dependent of L-Arginine in the renaturation buffer. With L-Arginine in the renaturation buffer an up to 20% higher yield was obtained. With increased residual NLS concentration no decrease in refolding yield was observed. All in one the renaturation yields were in the same range than for the NLG based renaturation of fusion protein 1.

Fusion Protein 2

[0091] The fusion protein used in this example (model protein 2) consists of 269 amino acids. The NPro-EDDIE-q15 moiety has 155 amino acids and the fusion partner consists of 102 amino acids. Based on the amino acid sequence the isoelectric points were calculated with 8.51 and 9.35 for the fusion protein and the fusion partner. The molecular masses were calculated with 17545.4 Da for Npro-EDDIEq15, 11232.8 Da for the fusion partner and 28760.5 Da for the fusion protein. In comparison to the naturally occurring variant of the fusion partner 33 amino acids are attached to its C-terminus. The sequence of the fusion partner contains 6 cysteines, which enable three disulfide bridges.

[0092] FIG. 5 shows the results for the renaturation of fusion protein 2 for the renaturation buffer and NLS concentration dependent renaturation.

[0093] The yield by using the renaturation buffer 4 is significant dependent of the residual NLS concentration in the renaturation batch. The overall yield raises from 50% at 0.2% (w/v) to 83% with 0.8% (w/v) residual NLS. In renaturation buffer no such dependence f the residual NLS concentration could be observed. Slightly lower yields were obtained at the two lowest determined NLS concentrations.

[0094] FIG. 6 shows the kinetics for the renaturation of fusion protein 2 at selected protein concentrations in the NLS and urea based renaturation in renaturation buffer 4.

[0095] The kinetics of the renaturation is showing three main influences for the renaturation. [0096] 1. The NLS based renaturation is at equal protein concentrations independent of the used residual concentration of NLS in the refolding batch [0097] 2. The NLS based renaturation has a high leverage dependence of the fusion protein concentration [0098] 3. The NLS based renaturation achieves at equal fusion protein concentrations significant higher yields compared to the urea based renaturation