WO2003027136A2

WO2003027136A2 - Protein production

Info

Publication number: WO2003027136A2
Application number: PCT/GB2002/004223
Authority: WO
Inventors: Edward Fahey; James Alexander Purvis; John William Stairmand
Original assignee: Accentus Plc
Priority date: 2001-09-26
Filing date: 2002-09-18
Publication date: 2003-04-03
Also published as: GB0123114D0; EP1432504A2; AU2002331948A1; WO2003027136A3

Abstract

Proteins may be made by genetically engineered microorganisms, the protein being stored in the form of inclusion bodies (IB). The proteins in the inclusion bodies are in an insoluble and inactive form. They may be dissolved using a solubilisation reagent (18), and the resulting solution diluted so that the proteins refold into the active form. The dilution is performed by passing the solution and a diluting liquid into a fluidic vortex mixer (20), the dimensions of the mixer and the flow rates being such that the residence time of the liquid in the mixer (20) is less than 1 second. The very efficient mixing reduces the proportion of the protein that re-aggregates, and reduces the required degree of dilution so as to increase the concentration of the active, correctly-folded protein.

Description

Protein Production

This invention relates to a method and an apparatus for obtaining solutions of proteins that are in their correctly folded state, that is to say having the correct structure or active secondary and/or tertiary structure.

A range of complex organic molecules can be synthesized in cells including bacterial, plant, fungal and mammalian cells. As an example, this synthesis can be carried out with bacterial cells in a fermentation process. The desired product molecules must then be recovered and purified from the cells. In particular, in the production of proteins from genetically engineered microorganisms, the cells may over-express the protein and create insoluble aggregates of the protein known as inclusion bodies. In a known process these inclusion bodies are isolated from other cell components by cell lysis, followed by, for example centrifugation or microfiltration. The proteins in the inclusion bodies are in an insoluble and inactive form, presumably because the molecules are tangled and/or incorrectly folded. A known process involves treating the inclusion bodies with a solubilisation reagent (so the molecules are no longer entangled) , and then removing or diluting the solubilisation reagent so that the proteins refold into the correctly-folded form. This dilution step tends to be inefficient, so that some of the protein reaggregates into an insoluble and inactive form, while the dilution required is usually very high, resulting in the formation of a large volume of very dilute protein solution. On an industrial scale the proportion of protein from the inclusion bodies that is converted into the correctly- folded product is typically only about 10%, and 20% conversion would be considered very good. The resulting correctly-folded protein may be an active protein that has a beneficial medicinal or pharmaceutical use.

According to the present invention there is provided a method for diluting a solution of solubilised protein, wherein the solution is intimately mixed with a dilution liquid by passage through a fluidic vortex mixer, the dimensions of the mixer and the flow rates being such that the residence time of the mixture in the mixer is less than 1 second.

A fluidic vortex mixer comprises a substantially cylindrical chamber with an axial outlet duct at the centre of an end wall of the chamber, and with at least one substantially tangential inlet near the periphery of the chamber to create a spiralling flow in the chamber. A second liquid is supplied through a second tangential inlet, or through a radial inlet. The chamber contains no turbulence-generating vanes or baffles. Such a mixer achieves a very intimate and thorough mixing of the two liquids in a very short time, but does not subject the liquids to high shear. It is also much less prone to being fouled, for example by proteinaceous deposits, than other types of mixer. The residence time means the time taken by the mixture to pass through the mixer.

As a consequence of this more efficient mixing, less of the protein undergoes aggregation, so more protein forms the active product. It can also be expected that less dilution is required than using prior art dilution techniques, so that the final concentration of the active, correctly-folded protein will be well above 0.01 mg/ml, for example it may be as high as 0.10 mg/ml or above . Examples of proteins are enzymes and hormones, and the term protein should also be construed as encompassing glycoproteins and lipoproteins or mixtures thereof. It will also be appreciated that the process may also be applied to a mixture of proteins, for example dilution liquid can be added to a solution of one or more proteins that has been obtained from more than one type of cell or to a solution of one or more proteins that has been obtained from different cells from the same class of organism. For example proteins obtained from two strains of yeast in a fermentation process or two types of bacteria in a fermentation process or a protein obtained from a yeast combined in solution with a protein obtained from a bacterium. Proteins may be present in solution as a monomer or dimer or consist of multiple sub-units.

The invention will now be further and more particularly described, by way of example only, and with reference to the accompanying drawing which shows a flow diagram for a protein recovery system.

Referring to the drawing, a procedure 10 is illustrated for obtaining active protein. Bacteria (Escherichia coli) genetically engineered to make the desired protein are grown 12 in a fermentation vessel.

The bacteria over-express the protein, which is stored as inclusion bodies within the bacteria in an insoluble, tangled and inactive state. When sufficient fermentation has taken place, the aqueous suspension of the cell is subjected to cell lysis 14 and then centrifugation 16 to separate the inclusion bodies IB from a waste stream. The inclusion bodies IB are then solubilised 18 by dissolution in a suitable reagent such as urea or guanidine hydrochloride, this causing the proteins to become disentangled. The resulting solution of inactive proteins is supplied to one tangential inlet 21 of a fluidic vortex mixer 20, refolding buffer (an aqueous solution containing a lower concentration of the solubilising reagent plus additional chemicals to assist the refolding process such as glutathione) as diluting agent is supplied to a second tangential inlet 22 of the mixer 20, so that a dilute solution emerges from an axial outlet 24 after a residence time in the mixer 20 typically less than 0.1 s. The flow rate of the refolding buffer may be 200 times greater than that of the solution of inactive proteins. By way of example, the cylindrical chamber in the mixer 20 might be of diameter 10 mm and of height 2 mm, each inlet duct 21 and 22 being of cross-sectional

2 area 1 mm , and the outlet duct 24 being of cross-

2 sectional area 2 mm ; with a flow rate of 1 litre per minute the residence time would be about 8 ms . If the flow rate through this mixer 20 were halved to 0.5 1/min the mean residence time would double to about 16 ms .

The outflow from the mixer 20 may be supplied to a holding tank 26 to allow more time for the protein to refold into the active form, preferably at least one hour. The holding tank can be a plug flow reactor, or a stirred reaction vessel with baffles. The plug flow reactor can be of the pulsed type, for example such as that described in WO 00/29545, and can be operated in a batch or continuous mode. The discharge from the vortex mixer would be fed directly into the pulsed plug flow reactor.

For the continuous mode a residence time of up to 5 hours is preferred so that a flow rate of 100 cm per minute (i.e. 6 litres per hour) would require a reactor volume of 30 litres. A residence period of 5 hours in the plug flow reactor would be expected to increase the refolding yield from around 10% to 30%. A continuous flow of low yield refolded proteins from the vortex mixer enters the plug flow reactor, which continuously discharges a flow of high yield refolded proteins. For the batch mode of operation a smaller plug flow reactor volume would be used compared to the continuous mode. For the batch mode the reactor would initially be empty and on start-up the vortex mixer would pump a solution of refolded proteins into the plug flow reactor, which would gradually fill. As soon as fluid entered the plug flow reactor, pulsations would be imposed. Once the plug flow reactor was full, all the feed to the vortex mixer would be removed and the plug flow reactor would be left pulsing and gently mixing the solution until the required 5 hour residence time had elapsed.

Whether operated continuously or batch-wise, the combination of the vortex mixer and the plug flow reactor would impose the desired mixing regimes of rapid initial mixing and gentle long residence time processing, both of which are required to attain high yields .

The solution may also be subjected to further processing such as liquid chromatography and filtration to separate any agglomerated (and therefore inactive) protein from the active protein in solution.

The contents of the holding tank may be subjected to irradiation by ultrasound by the use of transducers (not shown in Figure 1) to help prevent re-aggregation of proteins as refolding takes place. The ultrasonic treatment can be used in conjunction with a stirred tank and baffles. Refolded proteins, and in particular active proteins, produced in accordance with the present invention may be formulated into compositions that are useful for pharmaceutical applications for administration by various routes including oral, intravenous, subcutaneous, intramuscular, intraperitoneal routes. Compositions for administration may be in the form of powders, liquid, tablet or capsules. Compositions can contain one or more proteins that are present in solution before undergoing refolding or are refolded separately and then blended together.

Claims

1. A method for diluting a solution of solubilised protein, wherein the solution is intimately mixed with a dilution liquid by passage through a fluidic vortex mixer, the dimensions of the mixer and the flow rates being such that the residence time of the mixture in the mixer is less than 1 second.

2. A method as claimed in claim 1 wherein the residence time in the fluidic vortex mixture is less than 0.1 seconds .

3. A method as claimed in claim 1 or claim 2 wherein the solution emerging from the fluidic vortex mixture is supplied to a holding tank providing a residence time of at least 1 hour.

4. A method as claimed in claim 3 wherein the holding tank is a pulsed plug flow reactor.

5. A process for generating a solution of soluble protein in its correctly-folded form, in which solubilised protein is intimately mixed with a dilution liquid by passage through a fluidic vortex mixer, the dimensions of the mixer and the flow rates being such that the residence time of the mixture in the mixer is less than 1 second.

6. A solution of soluble protein produced by the process as claimed in claim 5 wherein the protein comprises enzymes, hormones, glycoproteins or lipoproteins or mixtures thereof.

7. A process as claimed in claim 5 in which the proteins are derived from bacterial, plant, fungal or mammalian cells.

8. A process as claimed in claim 7 in which the proteins are derived from different species of the same cell type