WO2003002590A2

WO2003002590A2 - A protein folding reactor

Info

Publication number: WO2003002590A2
Application number: PCT/GB2002/003017
Authority: WO
Inventors: Anton Middelberg; Chew Tin Lee; Malcolm Mackley; Mark Buswell
Original assignee: Cambridge University Technical Services Limited
Priority date: 2001-06-29
Filing date: 2002-06-28
Publication date: 2003-01-09
Also published as: WO2003002590A3; GB0116038D0; AU2002314356A1

Abstract

A protein folding reactor comprising: a first mixing chamber for holding a folding buffer; one or more of feeders for feeding denatured protein into the first mixing chamber, each feeder feeding at a respective feed point; and a first mixing mechanism capable of effecting high intensity mixing at each feed point in the first mixing chamber. The intensity of mixing adjacent each feed point is such that there is an increased yield of active protein when denatured protein is fed into the folding buffer.

Description

"A Protein Folding Reactor"

THE PRESENT INVENTION relates to a protein folding reactor and, in particular, a protein folding reactor in which a denatured protein is folded in a folding buffer.

It is known in the art to produce recombinant proteins in bacterial hosts such as E. coli. The problem with production of a protein in this way is that the protein is typically over-expressed in the bacteria and thus forms so-called "inclusion bodies" consisting of aggregations of the over-expressed protein together with various contaminants. The protein in the inclusion body is in an inactive form because it is incorrectly folded and thus has a non-functioning three- dimensional structure. Furthermore, there is the problem that the inclusion bodies are mixed with the other cell debris from the bacteria when they are recovered.

Accordingly, it is usual for recombinant proteins expressed in bacteria to undergo a series of processing steps in order to produce a purified, active protein. In particular, it is necessary for the protein in the inclusion bodies to be solubilised in a strong, denaturing chaotrope, such as 8M urea or GuHCl, typically providing a reducing environment. This results in the protein having a substantially random three-dimensioned structure which can then be renatured or "folded" into the active structure for that protein by dilution into a folding buffer containing a lower chaotrope concentration. The buffer usually provides an oxidising environment in order to promote disulphide bond formation in the protein. However, the refolding environment that is used is very specific to the particular protein, requiring a balance between an oxidising and reducing environment in order to form disulphide bonds appropriately.

While a variety of methods exist for industrial protein refolding, the most common method involves direct injection of the denatured (i.e. solubilised) protein solution into a stirred tank filled with a refolding buffer. The problem with this method, especially on an industrial scale, is that aggregation of inactive proteins occurs which leads to a low yield of active protein.

A typical prior art process for folding of a denatured protein is disclosed in US-A-4,933,434. This document discloses a process wherein a solution is prepared of the protein to be folded in a "critical concentration" in a folding buffer. However, the prior art does not appreciate the importance of the intensity of mixing of the protein in the folding buffer in order to avoid aggregation of the protein. Accordingly such prior art processes produce relatively low yields of active protein.

The present invention seeks to alleviate one or more of the above problems.

According to one aspect of the present invention, there is provided a protein folding reactor comprising: a first mixing chamber for holding a folding buffer; one or more of feeders for feeding denatured protein into the first mixing chamber, each feeder feeding at a respective feed point; and a first mixing mechanism capable of effecting high intensity mixing at each feed point in the first mixing chamber, the intensity of mixing adjacent each feed point being such that there is an increased yield of active protein when denatured protein is fed into the folding buffer. Conveniently, the protein folding reactor further comprises a delivery vessel for holding a solution of denatured protein, at least a portion of the delivery vessel being in fluid communication with the first mixing chamber, via the one or more feeders.

Preferably, the protein folding reactor further comprises means for urging the solution of denatured protein from the delivery vessel to the first mixing chamber.

Advantageously, the means for urging the solution of denatured protein from the delivery vessel to the first mixing chamber comprises means for pressurising the delivery vessel relative to the mixing chamber.

Conveniently the delivery vessel has a pressure of between 110 kPa and 400 kPa.

Preferably the portion of the delivery vessel in fluid communication with the first mixing chamber is located inside the first mixing chamber.

Advantageously the portion of the delivery vessel in fluid communication with the first mixing chamber comprises an elongate member, such that the delivery vessel is in fluid communication with the first mixing chamber along the length of the elongate member.

Conveniently the elongate member is located in the centre of the first mixing chamber. Preferably the protein folding reactor further comprises a pump for pumping the solution of denatured protein through the delivery vessel.

Advantageously the delivery vessel is in the form of a loop, the pump being capable of cycling the solution of denatured protein around the loop.

Conveniently the pump is capable of pumping the solution of denatured protein at a flow rate of between 10 and 50 times the rate of flow of the solution of denatured protein from the delivery vessel to the first mixing chamber.

Preferably the protein folding reactor further comprises: a second mixing chamber, in fluid communication with the first mixing chamber; and a second mixing mechanism for effecting mixing in the second mixing chamber.

Advantageously the first mixing mechanism is capable of effecting mixing at a greater mixing intensity than the second mixing mechanism.

Conveniently the second mixing mechanism and the second mixing chamber comprise a stirred tank reactor.

Preferably the first and second mixing chambers are in fluid communication via a re-circulating loop such that folding buffer is recirculatable between the first and second mixing chambers.

Advantageously the one or more feeders are located in the recirculating loop, upstream of the first mixing chamber.

Conveniently the first mixing mechanism comprises an oscillatory flow mixing unit. Although, it is to be understood that the first mixing mechanism could comprise an oscillating grid reactor, a standard stirred tank reactor, a static mixer or jet mixer, namely, any type of mixer capable of effecting the requisite high intensity mixing.

Preferably the oscillatory flow mixing unit comprises one or more baffles for increasing mixing in the oscillatory flow unit.

Advantageously the baffles are substantially annular.

Conveniently each of said one or more baffles are provided with a plurality of apertures.

Advantageously said one or more feeders comprise a plurality of feeders.

Preferably the plurality of feeders comprise a filter.

Advantageously the filter comprises a membrane.

Conveniently the membrane has a molecular weight cut off of between 100 and 500kDa.

Advantageously the intensity of mixing is equivalent to an oscillatory Reynolds number (Re₀) of at least 10, preferably at least 400 and more preferably at least 1500.

Preferably the intensity of mixing is equivalent to a stirred reactor Reynolds number (Re_st) of at least 100, preferably 1000, and more preferably 10000. Conveniently the intensity of mixing adjacent each feed point is characterised by a Kolmogorov Length Scale of less than 1mm, preferably less than 0.1 mm, and more preferably between 0.01 mm and 0.1 mm.

According to another aspect of the present invention there is provided a method of folding proteins comprising: feeding denatured protein into an oscillatory flow mixing unit containing folding buffer, and effecting a pulsatile motion through the oscillatory flow mixing unit to effect mixing of the denatured protein and the folding buffer.

According to another aspect of the present invention, there is provided a method of folding a protein comprising the steps of: determining the optimum intensity of mixing of the denatured protein in a folding buffer to provide the optimum yield of active protein; feeding the denatured protein into the folding buffer at one or more feed points; and mixing the denatured protein and the folding buffer at the optimum intensity adjacent the feed points.

Conveniently the denatured protein is in a denaturant and the step of determining the optimum intensity of mixing comprises determining the intensity of mixing at which the rate of removal of denaturant from the protein and the rate of dispersion of protein in the folding buffer provide the optimum yield of active protein.

Advantageously the intensity of mixing is equivalent to an oscillatory Reynolds number (Re₀) of at least 10, preferably at least 400 and more preferably at least 1500. Preferably the intensity of mixing is equivalent to a stirred reactor Reynolds number (Re_st) of at least 100, preferably 1000, and more preferably 10000.

Conveniently the intensity of mixing adjacent each feed point is characterised by a Kolmogorov Length Scale of less than 1mm, preferably less than 0.1 mm, and more preferably between 0.01 mm and 0.1 mm.

Preferably, the method uses the protein folding reactor described above.

Advantageously the protein folding reactor further comprises a delivery vessel, at least a portion of which is in fluid communication with the first mixing chamber and the step of feeding denatured protein into the first mixing chamber comprises the steps of: providing a denaturing buffer in the delivery vessel; and feeding a cell suspension containing the protein into the delivery vessel such that the protein is denatured.

In order that the invention may be more readily understood and so that further features thereof may be appreciated, embodiments thereof will now be described, by way of example, with reference to the accompanying drawings, in which:

Figure 1 is a cross-sectional view of a protein folding reactor in accordance with one embodiment of the present invention;

Figure 2 is a plan view of a portion of the protein folding reactor of Figure 1; Figure 3 is a plan view of a portion of the protein folding reactor in accordance with another embodiment of the invention;

Figure 4 is a schematic view of a protein folding reactor in accordance with another embodiment of the present invention;

Figure 5 is a cross-sectional view of a protein folding reactor used in Example l;

Figure 6 is a graph showing the results of Example 1, a comparison between the yield of active protein when denatured protein is mixed with refolding buffer at one and two feed points in an oscillatory flow reactor;

Figure 7 is a graph showing the results of Example 2, a comparison between the yield of active protein when denatured protein and folding buffer are mixed at different intensities;

Figure 8 is a plan view of a stirred - tank reactor used in a comparative experiment in Example 2;

Figure 9 is a cross - sectional view of the stirred - tank reactor shown in Figure 8;

Figure 10 is a cross - sectional view of another embodiment of a protein folding reactor according to the present invention;

Figure 11 is a plan view of a portion of the reactor of Figure 10; and Figure 12 is a graph showing the results of Example 3, which exemplifies the relationship between the yield of active protein obtained on varying the intensity of mixing.

Referring to Figure 1, a protein folding reactor 1 is shown, in accordance with one embodiment of the present invention. The protein folding reactor 1 comprises a recirculation loop 2 consisting of a ceramic, elongate, tubular membrane 3 connected at either end to a recirculation pipe 4. The tubular membrane 3 is 25cm in length and has a circular cross-section with a diameter of 0.7cm. Accordingly, the tubular membrane 3 has a filtration area of approximately 50cm . The membrane has a molecular weight cut-off of 300kDa. However, in other embodiments of the invention, the molecular weight cut-off varies, depending on the nature of the protein being folded, and is typically in the region of 100 to 500 kDa, more preferably 200 to 400 kDa. In certain embodiments, a micro filtration membrane with a nominal 0.1 to 0.45 micron pore size is provided.

A holding tank 5 is provided in the recirculation pipe 4 for holding a solution of denatured or solubilised protein, together with denaturant.

The recirculation pipe 4 is substantially "C" -shaped, with the end of either arm of the "C" making a fluid-tight connection to either end of the tubular membrane 3. The recirculation loop 2 thus contains the solution denatured protein. The solution is enclosed within the recirculation loop 2, except for the tubular membrane 3, out of which it may pass. As such, the recirculation loop 2 acts as a delivery vessel, holding the solution of denatured protein 5. A peristaltic pump 6 is provided in a portion of the recirculation pipe 4 in order to drive the solution of denatured protein around the recirculation loop 2 at a rate of between 5 and 50ml per hour.

A pressure valve 7 is provided in another portion of the recirculation pipe 4, such that the tubular membrane 3 is located between the peristaltic pump 6 and the pressure valve 7. The pressure valve 7 stops rupture or damage to the recirculation loop 2, particularly the tubular membrane 3 caused, for example, by a blockage in the loop 2. The pressure valve 7 provides a back pressure to allow variation of pressure in the elongate tubular membrane 3 and hence variation in the rate of delivery of denatured protein through the membrane 3. Thus the solution of denatured protein is maintained at a pressure of between HOkPa to 400kPa (0.1 and 3 barg) within the elongate tubular membrane 3 by the action of the peristaltic pump 6 and the pressure valve 7.

The protein folding reactor 1 also comprises an oscillatory flow mixing unit 8. An oscillatory flow mixing unit is a device which mixes the fluid held within it by providing oscillating pressure waves in the fluid. The oscillatory flow mixing unit 8 comprises an elongate tubular mixing vessel 9 having a length of 29.5cm and a circular cross-section with an outer diameter of 3.2cm and a wall thickness of 0.3cm. The tubular membrane 3 is located inside and coaxial with the mixing vessel 9 with each arm of the recirculation pipe 4 extending via fluid tight seals 10, 11 through the wall of the mixing vessel 9. Thus the remainder of the recirculation pipe 4 is outside the mixing vessel 9. First and second supports 12, 13 are provided at either end of the tubular membrane 3, opposite the respective arms of the recirculation pipe 2, connecting either end of the tubular membrane 3 to the inner surface of the mixing vessel 9, in order to support the tubular membrane 3. The mixing vessel 9 contains a protein folding buffer 14 and is at atmospheric pressure. In some other embodiments, a nitrogen blanket _. is provided in the mixing vessel 9 at a pressure slightly higher than atmospheric pressure but in these embodiments the pressure in the mixing vessel 9 is still less than the pressure in the tubular membrane 3.

At one end of the mixing vessel 9, a reciprocating piston 15 is provided, connected to an oscillator drive 16. One end of the piston 15 extends into the mixing vessel 9 via a seal 17. The oscillator drive 16 can cause the piston 15 to reciprocate relative to the mixing vessel 9 in the direction of the longitudinal axis of the mixing vessel 9.

Referring also now to Figure 2, a series of seven annular baffles 18 are fixed by arms 40 extending from their outer edges to the interior of the mixing vessel 9, coaxial with the mixing vessel 9 and the tubular membrane 3. Each baffle 18 is separated by a distance of 3.5cm. In other embodiments of the invention, in which a different size of protein folding reactor 1 is provided, the baffles are separated by a distance of between 1 and 1.5 times the outer diameter of the mixing vessel 9. There is a gap of 0.5 mm between the outer perimeter 19 of each of the annular baffles 18 and the interior of the mixing vessel 9 and thus the outer diameter of the baffles is 3.1cm. The central aperture 20 in each baffle, through which the tubular membrane 3 extends, has a diameter of 1.82cm.

In the other end of the mixing vessel 9, opposite the piston 15, a head space 21 is provided as an extension to the mixing vessel 9. The head space has a length of 8cm. In use, denatured protein is fed into the holding tank 5 to deliver a predetermined quantity of denatured protein into the recirculation loop 2. Alternatively, denatured protein can be added through a feeder arrangement in the peristaltic pump 6. The peristaltic pump 6 drives the solution of denatured protein around the recirculation loop 2 in the direction of the arrows 22 at a rate of between 5 and 50 ml/hour. In some embodiments, the peristaltic pump 6 drives the solution of denatured protein in the opposite direction. Thus the solution of denatured protein cycles around the recirculation loop 2, passing through the circulation pipe 4, into and along the tubular membrane 3 and then back along the recirculation pipe 4, before repeating its journey.

Because the recirculation loop 2 is maintained at a higher pressure than the interior of the mixing vessel 9 some of the solution of denatured protein passes out of the re-circulation loop 2, through the tubular membrane 3, into the mixing vessel 9, as the solution of denatured protein cycles around the recirculation loop 2. Thus the pressurisation of the recirculation loop 2 acts as a means for urging the solution of denatured protein from the recirculation loop 2 to the mixing vessel 9. Any contaminants in the denatured protein solution, such as cell debris, that are larger than the molecular weight cut-off of the membrane 3 are prevented from passing out of the tubular membrane 3, into the mixing vessel 9. Accordingly, each perforation in the tubular membrane 3 acts as a feeder for feeding denatured protein into the mixing vessel 9. Furthermore, the tubular membrane 3 acts as a filter between the recirculation loop 2 and the mixing vessel 9. Because the filtration area of the tubular membrane 3 is relatively large, the solution of denatured protein 5 is widely dispersed throughout the mixing vessel 9. This improves the yield of folded protein by reducing problems associated with inefficient dispersion at a large scale as is explained in greater detail below. In the meantime, the oscillator drive 16 drives the piston 15 through the seal 17 to provide a pulsatile motion in the folding buffer 14. The piston 15 reciprocates at a frequency of between 0.5 and 8Hz and thus drives the folding buffer 14 in a pulsatile motion in the direction of the arrow 23, i.e. in the direction of the longitudinal axis of the mixing vessel 9. The amplitude of the oscillation is between 1 and 8mm. Each pair of adjacent baffles 18, together with the respective ends of the mixing vessel 9, effectively creates a series of eight chambers, which cause turbulence in the pulsatile motion of the folding buffer 14. This ensures that there is a mixing flow in both the axial and radial directions. Furthermore, because denatured protein passes through the tubular membrane 3 along its length, denatured protein is fed to each chamber in the mixing vessel 9. Thus very effective mixing of the solution of denatured protein in the folding buffer 14 takes place and there is rapid dispersion of the solution of denatured protein in the folding buffer 14 even in embodiments of the invention carried out on a larger scale. In particular, there is high intensity mixing of the denatured protein in the folding buffer adjacent each feed point where the protein feeds into the folding buffer.

The denatured protein folds into an active protein as it is mixed into the folding buffer 14. Although some of the denaturant in the solution of denatured protein also passes from the tubular membrane 3 into the mixing vessel 9, it is diluted in the folding buffer and so the overall effect of the mixture in the mixing vessel 9 is to cause the folding of the protein.

It is to be appreciated that, because the solution of denatured protein is fed into the mixing vessel 9 along the length of the tubular membrane 3 and because of the very effective mixing of the denatured protein in the folding buffer 14, aggregation of protein in the mixing vessel 9 is greatly reduced and thus high yields of folded protein are obtainable. In addition, because the solution of denatured protein is constantly circulated around the recirculation loop 2, caking on the surface of the tubular membrane is avoided. Furthermore, the tubular membrane 3 prevents any residual particulate material from entering and contaminating the mixing vessel 9, removing the need for a separate filtration step prior to refolding.

In this embodiment of the invention, after a predetermined quantity of protein has been folded in the mixing vessel 9, the contents of the mixing vessel 9 are removed for further purification steps in which the active folded protein is removed from the folding buffer 14. In some other embodiments of the invention, the contents of the mixing vessel 9 are continuously removed from the mixing vessel 9 for purification as fresh folding buffer 14 is simultaneously added to the mixing vessel 9. In these embodiments, the pulsatile motion of the folding buffer 14 is superimposed on a continuous flow of the folding buffer 14 through the mixing vessel 9. Thus, there is a net flow of folding buffer 14 through the mixing vessel 9. These embodiments are particularly appropriate in connection with those embodiments of the invention, described above, in which denatured protein is continuously added to the recirculation loop 2.

In other embodiments of the invention, the rate of flow of the solution of denatured protein is different from that described above. In these embodiments, it is preferable that the rate of recirculation flow of the solution of denatured protein be between approximately 10 and 50 times its rate of flow from the tubular membrane 3 into the mixing vessel 9. However, the yield of refolded protein is relatively unchanged by the value selected within this range. Referring now to Figure 3, in some embodiments of the invention each annular baffle 18 additionally comprises a plurality of further apertures 24, at varying locations around the annulus. In such embodiments, the central aperture 20 of each baffle 18 has a reduced diameter to promote shear stresses near the tubular membrane 3. Thus, improved mixing in the mixing vessel 9 occurs.

In some embodiments, denatured protein, itself, is not fed into the recirculation loop 2. Instead a cell suspension is fed into the recirculation loop 2 from the holding tank 5. An extracting chaotrope is provided in the recirculation loop 2 which denatures the protein in the cell suspension. The tubular membrane 3 prevents the residual particulate material from the cell suspension from entering the mixing vessel 9. Thus the protein folding reactor 1 carries out the steps of extracting protein, denaturing it and folding the protein integrally.

In some other embodiments of the invention, the recirculation loop 2 is omitted from the protein folding reactor 1. In these embodiments, the solution of denatured protein is fed directly into the mixing vessel 9 at one or more injection points, without an intervening membrane.

Reference will now be made to Figure 4 in which an alternative embodiment of the invention is shown schematically. A protein folding reactor 1 comprises an oscillatory flow mixing unit 8 which is substantially the same as the oscillatory flow mixing unit described previously. Extending from a first end 25 of the oscillatory flow mixing unit 8 is a recirculation pipe 26 which leads to a stirred - tank reactor 27. The stirred tank reactor 27 is substantially larger than the oscillatory flow mixing unit 8 and comprises a stirrer 28 which can mix the contents of the stirred tank reactor 27. Leading away from the stirred tank reactor 27, the recirculation pipe 26 continues, via a pump 27 to connect back to a second end 28 of the oscillatory flow mixing unit 8. Thus the oscillatory flow mixing unit 8 and the stirred tank reactor 27 are connected by the recirculation pipe 26 to form a loop. Adjacent the connection of the recirculation pipe 26 to the second end 28 of the oscillatory flow mixing unit 8 a feeder pipe 29 connects to the recirculation pipe 26. The feeder pipe 29 leads from a storage tank 30 that contains a solution of denatured protein.

In use of this alternative embodiment, a solution of folding buffer is provided in the oscillatory flow mixing unit 8, the stirred tank reactor 27 and the recirculation pipe 26. The solution is driven around the loop in the direction of the arrows 31 by the pump 27. A solution of denatured protein is fed from the tank 30, through the feeder pipe 29 into the recirculation pipe 26, just upstream of the oscillatory flow mixing unit 8. Thus the solution of denatured protein then enters the second end 28 of the oscillatory flow mixing unit 8. In the oscillatory flow mixing unit 8, a pulsatile motion of the liquid (i.e. the folding buffer and the solution of denatured protein) is effected, as is explained in more detail in relation to previous embodiments. Therefore the solution of denatured protein is very effectively mixed with the folding buffer.

After a period of between 1 and 100 seconds, the flow of liquid around the loop is such that the mixture of denatured protein and folding buffer is moved from the second end 28 to the first end 25 of the oscillatory flow mixing unit 8. From there the mixture moves, via the recirculation pipe 26 to the stirred tank reactor 27. In the stirred tank reactor 27, the denatured protein and folding buffer are further mixed by the stirrer 28. Subsequently, the mixture of denatured protein and the folding buffer are pumped via the recirculation pipe 26 and the pump 27 back into the second end 28 of the oscillatory flow mixing unit 8 and the process is repeated. After a predetermined quantity of protein has been folded, the contents of the protein folding reactor 1 are removed for further purification steps in which the active folded protein is removed from the folding buffer. It has been found that in order to obtain increased yields of active protein, it is only necessary for high intensity of mixing of the denatured protein and the refolding buffer to take place for a period of between 1 and 100 seconds. Therefore this embodiment provides this initial high intensity mixing in the oscillatory flow mixing unit 8, while extended mixing occurs in a standard, low - intensity, stirred-tank reactor. This embodiment allows for more economic production of active, folded protein because a standard stirred - tank reactor is generally cheaper than an oscillatory flow mixing unit of the same size. Therefore a greater quantity of denatured protein can be folded at any one time using this embodiment than using an oscillatory flow mixing unit, of equivalent cost, alone.

In a preferred embodiment, the solution of denatured protein may be added directly into the oscillatory flow mixing unit but is, in any case, fed into the protein folding reactor 1 such that it is initially mixed in the oscillatory flow mixing unit 8.

In variations of this alternative embodiment, the oscillatory flow mixing unit 8 is replaced by another high intensity mixing unit.

When denatured protein is fed into folding buffer in a reactor the mixing that occurs is complex. However, in essence, when a denatured protein solution is fed into a folding buffer, the denatured solution is very rapidly "sheared" into discrete elements that can be said to move within the folding buffer. The denaturant and the protein diffuse from the discrete elements into the folding buffer at different rates. Typically, the protein diffuses into the refolding buffer with a time constant of between 0.01 sec and 100 sec when the size of the discrete elements is between approximately 0.01 mm and 1 mm, respectively. However, these time constants vary depending on the size of the protein being folded and the properties of the solution. In contrast, the denaturant diffuses into the refolding buffer at a faster rate (typically at a rate approximately ten times the rate of diffusion of the protein) such that the discrete elements become depleted of denaturant before the protein has fully diffused into the refolding buffer. In these circumstances, aggregation of the protein tends to occur instead of correct folding of the protein.

A small discrete element size is generated in high turbulence (i.e. high intensity mixing) because the protein need only diffuse a short distance into the refolding buffer. This causes rapid concentration uniformity of the mixture at the microscale of the discrete elements. The lower the intensity of mixing, the higher the concentration of protein remains in the discrete elements for a longer period of time resulting in more aggregation of protein and a lower yield of active protein. This is because the lower the intensity of mixing the greater the distance that protein must diffuse into the refolding buffer.

Conversely, aggregation of protein tends to be avoided and an increased yield of active protein produced when the distance for diffusion of protein is shorter. This is achieved by increasing the intensity of mixing of the denatured protein in the folding buffer.

It is to be understood that in order to obtain an even higher yield of active protein, concentration uniformity of protein in the folding buffer must be rapidly obtained at the scale of the reactor (i.e. the macroscale) as well as at the scale of the discrete elements (the microscale). When this is achieved, the protein can diffuse from the discrete elements into fluid which is rich in folding buffer and lean in protein. This reduces the total protein concentration and aggregation of the protein thus tends to be avoided. Obtaining rapid concentration uniformity of protein on a macroscale is achieved by feeding the denatured protein widely throughout the reactor using a plurality of feed points and by intense and uniform mixing to ensure further rapid dispersion of the discrete elements throughout the reactor

It is also the case that different proteins are characterised by having different "energy landscapes" and hence have different folding behaviours. Depending on this energy landscape, a particular protein will have an optimal rate of denaturant removal which maximises the yield of active protein.

Accordingly, in order to obtain an optimum yield of active protein, there are two competing considerations. Firstly, there is the requirement to have the highest possible mixing intensity in order to ensure rapid attainment of concentration uniformity at both the microscale and macroscale. As is explained above, the attainment of macroscale uniformity is facilitated by feeding of the denatured solution throughout the reactor by using a plurality of feed points, and by rapidly dispersing the discrete elements throughout the reactor by high intensity mixing. The attainment of microscale uniformity is facilitated by decreasing the distance for diffusion and hence the size of the discrete elements, again by high intensity mixing. Secondly, there is the requirement that the removal of denaturant from the protein not be too rapid as this can result in unfavourable folding behaviour due to the energy landscape of the protein. Since the energy landscape of a protein is specific to that protein, these considerations will differ from protein to protein and, accordingly, the optimum mixing intensity will differ from protein to protein. Furthermore, it is impossible to define the optimum mixing intensity of a protein a priori and this must be determined empirically. Mixing intensity on the microscale (i.e. the scale of the discrete elements of protein and denaturant in the folding buffer) can be defined by the Kolmogorov Length Scale (KLS) as follows:

KLS ^V

\ ε j where ε is the energy density during mixing (W/kg) and υ is the kinematic viscosity (m /s). The Kolmogorov Length Scale can be used to define the mixing of denatured protein adjacent to the feed points where it is fed into folding buffer.

Mixing intensity on the macroscale (i.e. over an entire reactor) can be quantitatively defined by a dimensionless group called the Reynolds number. The Reynolds number is calculated as follows:

where p = density of a fluid travelling at velocity v in a pipe of diameter D. A high Reynolds number corresponds to a high average level of turbulence.

In a stirred reactor, the definition of the Reynolds number (Re_st) is as follows:

where D_a = impeller diameter (m); p = fluid density (kg/m³); μ = viscosity (kg/m.s) and N = rotational speed of impeller (revolutions/sec). In an oscillatory flow mixing reactor, a different measure of mixing intensity is used: the oscillatory Reynolds number (Re₀), which is defined as follows.

Dωχ_o

Re₀ =

where D is the tube diameter (m), ω is the angular frequency of the oscillator drive (rad.s^"1), XQ is the oscillatory amplitude measured from centre-to-peak (mm), and v is the kinematic viscosity (m s^" ) (Mackley MR. 1991. Process innovation using oscillatory flow within baffled tubes. Trans IChemE, Part A 59:197-199).

It is to be appreciated that the Reynolds numbers for mixing in reactors of different geometries (such as a stirred reactor and an oscillatory flow mixing reactor) are not directly comparable. For example, a Re_st of 725 for a stirred reactor would generally be considered to be relatively low intensity mixing whereas a Re₀ of 725 for an oscillatory flow mixing reactor would generally be considered to be relatively high intensity mixing.

In order to determine the optimum mixing intensity for a particular protein, a series of experiments are carried out, in which the mixing intensity of the denatured protein in the folding buffer is varied and the yield of active protein is measured. Example 1 discusses one possible protocol for determining the yield of active protein when denatured protein is fed into folding buffer and mixed at different intensities. The yield of active protein produced by each experiment is compared and the results interpolated in order to determine the optimum mixing intensity at which the highest yield of active protein is produced. If the experiment conducted with the highest mixing intensity also provides the highest yield of active protein then further experiments are conducted with still higher mixing intensities until the yield of active protein diminishes. Once the approximate value of the optimum mixing intensity has been determined, a further series of experiments with smaller variations in mixing intensity around the approximate value can be conducted in order to provide a more accurate figure for the optimum mixing intensity.

It is to be appreciated that, as used herein, the term "optimum mixing intensity" includes, within certain embodiments of the invention, the range of mixing intensities at which yields of active protein within 5%, 10 % or 20% of the highest achievable yield of active protein are obtained.

It has been determined that the KLS of the optimum mixing intensity adjacent to the feed points when denatured protein is fed into folding buffer is, in some embodiments, less than 1mm, preferably less than 0.1mm and more preferably between 0.1mm and 0.01 mm.

It has also been determined that the Reynolds number of the optimum mixing intensity for proteins in an oscillatory flow mixing reactor (i.e. Re₀) is, in some embodiments, not less than 10, is typically at least 400 and is often at least 1,500. The Reynolds number of the optimum mixing intensity for proteins in a stirred tank reactor (i.e. Re_st) is, in some embodiments, at least 100, preferably at least 1000 and more preferably at least 10000.

It is also to be appreciated that in order to reduce aggregation of protein when denatured protein is fed into a folding buffer, it is important that the optimum mixing intensity is effected adjacent the positions in the folding buffer into which the denatured protein is fed. This is because it is the initial dispersion of denatured protein in the folding buffer that must be controlled in order to avoid aggregation and increase the yield of active protein produced. It is preferred that the optimal mixing intensity be provided uniformly throughout the reactor in which the denatured protein and folding buffer are mixed.

Examples

Example 1

The oscillatory flow reactor having a dual-feed system and shown schematically in Figure 5 was used in Example 1. The oscillatory flow reactor 32 comprises a holding tank 33 for holding a solution of denatured protein. Leading from the tank 33 is a delivery pipe 34 which leads, via a peristaltic pump 35 to a point 36 at which the delivery pipe 34 bifurcates into first and second feeding lines 37 and 38. The first and second feeding lines 37 and 38 pass into the top of an oscillatory flow mixing unit 39 which is substantially the same as the oscillatory flow mixing unit described above, except that no recirculation loop 2 is provided and, in particular, no tubular membrane 3 is provided. Furthermore, each baffle has a central aperture 20 with a diameter of 12mm and an outer perimeter of 24mm. No further apertures 24 are provided in the baffles 18 and the outer perimeter 19 of each baffle 18 is flush with the interior of the vessel 9. The first feeding line 37 extends until the region in the oscillatory flow mixing unit 39 between the second and third baffles 18 and thus delivers the solution of denatured protein into the third chamber from the end of the oscillatory flow mixing unit 39. The second feeding line 38 until the region in the oscillatory flow mixing unit 39 between the fifth and sixth baffles 18 and therefore feeds into the sixth chamber from the end of the oscillatory flow mixing unit 39. Thus in use, the solution of denatured protein is pumped by the peristaltic pump 35 into the first and second feeding lines 37 and 38 and from there into the third and sixth chambers of the oscillatory flow mixing unit 39.

Denatured Lysozyme (15mg/ml Lysozyme, 8M urea, 32 mM DTT) was fed into the oscillatory flow mixing unit 34 at a total flow rate of 0.09 ml/min for 120 minutes using the peristaltic pump 35, to give a final protein concentration of lmg/ml. The initial volume of refolding buffer (4mM GSSG, 50 mM Tris-HCl, 1 mM EDTA, pH 8, 20°C) was 140 ml. Refolding was conducted under intense oscillation giving Re₀ = 1580 (frequency = 3.5 Hz, amplitude = 3 mm) and mild oscillation giving Re₀ = 250 (frequency = 0.65 Hz, amplitude = 1mm). The corresponding experiments at low and high mixing were denoted as Re_o250 (two points of feeding) and Re_o1580 (two points of feeding), respectively. Upon completion of feeding, the solution was left for 3 hours and oscillated at the selected mixing intensity. Samples were analyzed using RP-HPLC method.

The experiments were repeated using the single-feed oscillatory flow reactor described in relation to Example 2. In these comparative experiments, the flow rate was maintained at 0.09 ml/min, but this total flow was fed through one feed point instead of two feed points. The corresponding experiments at low and high mixing were denoted as Re_o250 and Re_o1580, respectively.

The results of the Example 1 are shown in Figure 6. As can be seen, the yield of active protein produced was greater if two feeding points for feeding denatured protein were provided than if only one feeding point was provided. Furthermore, the yield of active protein produced was greater if high intensity mixing of the denatured protein in the folding buffer took place than if relatively low intensity mixing took place.

Example 2

In this example a single-feed oscillatory flow reactor was used. The reactor is substantially the same as the reactor used in Example 1 except that the delivery pipe 36 does not bifurcate and leads to a single feeding line which extends until the region of the oscillatory flow mixing unit 39 between the third and fourth baffles 18, thus delivering denatured protein into the fourth chamber from the end of the oscillatory flow mixing unit 39.

As a comparative example, a standard stirred tank reactor was also used to mix denatured protein with folding buffer. Referring to Figures 8 and 9, the standard stirred - tank reactor 41 comprises a cylindrical vessel 42 having a diameter 43 of 63mm. The height 44 of the vessel 42 is 70mm. Three elongated fins 45 are located equidistant around the interior perimeter of the vessel 42, parallel to the longitudinal axis of the vessel 42. Each fin 45 has a width 46 of 6mm. Suspended along the longitudinal axes of the stirred - tank reactor 41 is an axle 47, the end of which is disposed 12mm from the end of the vessel 42. Extending radially from the end of the axle 47 are four paddles 48 in the form of a cross. Each paddle 48 has a length 49 of 9mm and a height 50 of 6mm. The paddles 48 are displaced radially from the axle 47 so that the overall distance 51 between the tips of opposing paddles is 33mm.

8M urea-denatured Lysozyme (15 mg/ml Lysozyme, 8M urea, 32 mM DTT) was fed into the oscillatory flow mixing unit 39 or stirred tank reactor at a flow rate of 0.09 ml/min for 120 minutes using a peristaltic pump, to give a final protein concentration in the reactor of 1 mg/ml. The initial volume of refolding buffer (4 mM GSSG, 50 mM Tris-HCl, 1 mM EDTA, pH 8, 20°C) was 140 ml. In the oscillatory flow mixing unit 39 refolding was conducted under intense oscillation giving Re₀ = 1580 (frequency = 3.5 Hz, amplitude = 3 mm) and mild oscillation giving Re₀ = 250 (frequency = 0.65 Hz, amplitude = 1 mm). In the stirred tank reactor, refolding was conducted at a mixing intensity of 40 rpm, providing Re_st = 725. Upon completion of feeding, the solution was left for 3 hours and mixed at the selected mixing intensity. Samples were analyzed using RP-HPLC method. The results of Example 2 are shown in Figure 7. A can be seen from Figure 7, the yield of active protein produced when the oscillatory flow mixing reactor was mixing at high intensity was significantly greater than the yield produced either by the oscillatory flow mixing unit at relatively low intensity mixing or by the standard stirred tank reactor.

Example 3

The aim of this example is to confirm that the Kolmogorov Length Scale resulting from turbulent mixing affects refolding yield. One of the difficulties of analysing mixing effects in stirred reactors is obtaining reproducible and well-defined mixing conditions. In order to overcome this problem an oscillating grid reactor was designed. An oscillating grid reactor provides a well-characterised hydrodynamic environment, thereby overcoming the difficulty of defining a single turbulent length-scale in a stirred-tank reactor. By altering the frequency of oscillation, the turbulent length-scale at the point of dilution within the reactor can be varied and related to the refolding yield. The magnitude of the Kolmogorov length-scale can be estimated from scaling arguments that balance the turbulent energy flux with the characteristic viscous dissipation (see A.M. Buswell, PhD Thesis, University of Cambridge 2002). These scaling arguments were used to provide an estimate of the turbulent length-scale at the point of dilution at various grid oscillation frequencies. These estimates are shown in Table 3-1 below. Table 3-1: Kolmogorov Length Scale for varying grid oscillation frequencies.

As illustrated in Figure 10, the oscillating grid 1 reactor includes a rectangular vessel 8 constructed of Perspex. A stainless steel grid 60 is oscillated in the vertical plane near the base of the vessel 8 by an eccentric drive shaft. The frequency, f, and stroke, St, of the grid oscillations can be varied between 2-10Hz and 0.5-1.5cm, respectively.

As shown in Figure 11, which illustrates the geometry of the oscillating grid, the grid 60 is fully defined by the width of the bars or rods 61 (d= 1.6mm) making up the grid and the grid mesh spacing (M=0.76cm). These two parameters are used to calculate the solidity of the grid 60 (defined as the ratio of closed area to total area), which is an important scaling variable as it affects the nature of the turbulence generated (see A.M. Buswell, PhD Thesis, University of Cambridge 2002). The solidity should be smaller than 40% for the wakes or jets generated above the bars to be stable. A peristaltic pump (not illustrated) was used to feed denatured-reduced lysozyme, via a 0.3175 cm 1/8" steel needle 63, into the vessel 8 containing refolding buffer. The depth of the needle 63 was adjusted such that denatured protein was injected just above the maximum stroke of the grid 60 where turbulence will be at its maximum and the turbulent length-scale can be estimated from scaling arguments.

Refolding was conducted by feeding 33.3ml of denatured-reduced lysozyme (9.6mg/ml lysozyme, 8M urea, 32mMDTT, 50mM Tris, lmM EDTA, pH 8.0, 37 °C) at 1.82ml/min for 18.3min into 500ml of refolding buffer (5.33mM GSSG, 50mM Tris, lmM EDTA, pH 8.0, «20 °C). The grid oscillation stroke was set to 1.3 cm and the frequency of oscillation was varied between 2 and 10Hz. After 3 hours the grid oscillation was stopped and a sample of the refolding solution taken. The sample was incubated overnight to ensure complete refolding. The sample was then acidified by addition of lOOμl of 10% TFA to 900μl of sample. The acidified sample was centrifuged and analysed by RP-HPLC. The final lysozyme concentration in the refolding buffer was 0.64mg/ml.

As shown in Figure 12, the yield of active lysozyme increased with increasing oscillating frequency up to 8Hz. At oscillating frequencies above 8Hz the yield decreased with increasing grid frequency. Therefore, the intensity of mixing and hence, the Kohnogorov Length Scale is proved to affect the yield of refolding.

The oscillating frequency can be related to the characteristic mixing length- scale via the calculated results presented in Table 3-1 above. A comparison of Table 3-1 and Figure 12 confirms that, for this particular reactor and protein, optimal mixing occurs at a Kolmogorov Length Scale of 50 micrometers (i.e., 0.05 mm).

As will be understood from the above description, there is synergy in the provision of a protein folding reactor that has plurality of feeding points for feeding denatured protein into folding buffer and that mixes the denatured protein and folding buffer at high intensity, leading to an increased yield of active protein. In contrast to the prior art, which teaches that aggregation of inactive protein occurs within the reactor bulk, it is thought that this synergy exists because aggregation in fact occurs within the dispersion zone where denatured protein is first introduced into the folding buffer. Furthermore, it is thought that relatively low yields of active protein occur when refolding is carried out on a large scale in prior art reactors because the initial dispersion of denatured protein into such reactors is inefficient due to mixing imperfections in the reactors. It is therefore important that high intensity mixing occurs when denatured protein is fed into folding buffer, which can be achieved, even on an industrial scale, using an oscillatory flow mixing reactor.

In the present specification "comprise" means "includes or consists of and "comprising" means "including or consisting of.

The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

Claims

1. A protein folding reactor comprising: a first mixing chamber for holding a folding buffer; one or more of feeders for feeding denatured protein into the first mixing chamber, each feeder feeding at a respective feed point; and a first mixing mechanism capable of effecting high intensity mixing at each feed point in the first mixing chamber, the intensity of mixing adjacent each feed point being such that there is an increased yield of active protein when denatured protein is fed into the folding buffer.

2. A protein folding reactor according to Claim 1, further comprising a delivery vessel for holding a solution of denatured protein, at least a portion of the delivery vessel being in fluid communication with the first mixing chamber, via the one or more feeders.

3. A protein folding reactor according to Claim 1 or 2 further comprising means for urging the solution of denatured protein from the delivery vessel to the first mixing chamber.

4. A protein folding reactor according to Claim 3 wherein the means for urging the solution of denatured protein from the delivery vessel to the first mixing chamber comprises means for pressurising the delivery vessel relative to the mixing chamber.

5. A protein folding reactor according to Claim 4 wherein the delivery vessel has a pressure of between 110 kPa and 400 kPa.

6. A protein folding reactor according to any one of Claims 2 to 5 wherein the portion of the delivery vessel in fluid communication with the first mixing chamber is located inside the first mixing chamber.

7. A protein folding reactor according to any one of Claims 2 to 6 wherein the portion of the delivery vessel in fluid communication with the first mixing chamber comprises an elongate member, such that the delivery vessel is in fluid communication with the first mixing chamber along the length of the elongate member.

8. A protein folding reactor according to Claim 7 wherein the elongate member is located in the centre of the first mixing chamber.

9. A protein folding reactor according to any one of Claims 2 to 8 wherein the protein folding reactor further comprises a pump for pumping the solution of denatured protein through the delivery vessel.

10. A protein folding reactor according to Claim 9 wherein the delivery vessel is in the form of a loop, the pump being capable of cycling the solution of denatured protein around the loop.

11. A protein folding reactor according to Claim 9 or 10 wherein the pump is capable of pumping the solution of denatured protein at a flow rate of between 10 and 50 times the rate of flow of the solution of denatured protein from the delivery vessel to the first mixing chamber.

12. A protein folding reactor according to any one of the preceding claims further comprising: a second mixing chamber, in fluid communication with the first mixing chamber; and a second mixing mechanism for effecting mixing in the second mixing chamber.

13. A protein folding reactor according to Claim 12 wherein the first mixing mechanism is capable of effecting mixing at a greater mixing intensity than the second mixing mechanism.

14. A protein folding reactor according to Claim 13 wherein the second mixing mechanism and the second mixing chamber comprise a stirred tank reactor.

15. A protein folding reactor according to Claim 14 wherein the first and second mixing chambers are in fluid communication via a re-circulating loop such that folding buffer is recirculatable between the first and second mixing chambers.

16. A protein folding reactor according to Claim 15 wherein the one or more feeders are located in the recirculating loop, upstream of the first mixing chamber.

17. A protein folding reactor according to any one of the preceding claims wherein the first mixing mechanism comprises an oscillatory flow mixing unit.

18. A protein folding reactor according to Claim 17 wherein the oscillatory flow mixing unit comprises one or more baffles for increasing mixing in the oscillatory flow unit.

19. A protein folding reactor according to Claim 18 wherein the baffles are substantially annular.

20. A protein folding reactor according to Claim 18 or 19 wherein each of said one or more baffles are provided with a plurality of apertures.

21. A protein folding reactor according to any one of the preceding claims wherein said one or more feeders comprise a plurality of feeders.

22. A protein folding reactor according to Claim 21 wherein the plurality of feeders comprise a filter.

23. A protein folding reactor according to Claim 22 wherein the filter comprises a membrane.

24. A protein folding reactor according to Claim 24 wherein the membrane has a molecular weight cut off of between 100 and 500kDa.

25. A protein folding reactor according to any one of the preceding claims wherein the intensity of mixing is equivalent to an oscillatory Reynolds number (Re₀) of at least 10, preferably at least 400 and more preferably at least 1500.

26. A protein folding reactor according to any one of the preceding claims wherein the intensity of mixing is equivalent to a stirred reactor Reynolds number (Re_st) of at least 100, preferably 1000, and more preferably 10000.

27. A protein folding reactor according to any one of the preceding claims wherein the intensity of mixing adjacent each feed point is characterised by a Kolmogorov Length Scale of less than 1mm, preferably less than 0.1 mm, and more preferably between 0.01 mm and 0.1 mm.

28. A method of folding proteins comprising: feeding denatured protein into an oscillatory flow mixing unit containing folding buffer, and effecting a pulsatile motion through the oscillatory flow mixing unit to effect mixing of the denatured protein and the folding buffer.

29. A method of folding a protein comprising the steps of: determining the optimum intensity of mixing of the denatured protein in a folding buffer to provide the optimum yield of active protein; feeding the denatured protein into the folding buffer at one or more feed points; and mixing the denatured protein and the folding buffer at the optimum intensity adjacent the feed points.

30. A method according to Claim 29 wherein the denatured protein is in a denaturant and wherein the step of determining the optimum intensity of mixing comprises determining the intensity of mixing at which the rate of removal of denaturant from the protein and the rate of dispersion of protein in the folding buffer provide the optimum yield of active protein.

31. A method according to Claim 29 or 30 wherein the intensity of mixing is equivalent to an oscillatory Reynolds number (Re₀) of at least 10, preferably at least 400 and more preferably at least 1500.

32. A method according to any one of Claims 29 to 31 wherein the intensity of mixing is equivalent to a stirred reactor Reynolds number (Re_st) of at least 100, preferably 1000, and more preferably 10000.

33. A method according to any one of Claims 29 to 32 wherein the intensity of mixing adjacent each feed point is characterised by a Kolmogorov Length Scale of less than 1mm, preferably less than 0.1 mm, and more preferably between 0.01 mm and 0.1 mm.

34. A method according to any one of Claims 28 to 33 using the protein folding reactor of any one of Claims 1 to 27.

35. A method according to Claim 34 wherein the protein folding reactor further comprises a delivery vessel, at least a portion of which is in fluid communication with the first mixing chamber and wherein the step of feeding denatured protein into the first mixing chamber comprises the steps of: providing a denaturing buffer in the delivery vessel; and feeding a cell suspension containing the protein into the delivery vessel such that the protein is denatured.

36. A protein folding reactor substantially as herein described with reference to and as shown in the accompanying drawings.

37. A method of folding proteins substantially as herein described with reference to the accompanying drawings.

38. Any novel feature or combination of features disclosed herein.