Patent Application
20100093982
The present application is related to the field of devices and methods for the precipitation of peptides and proteins.
In the course of the isolation of peptides and proteins, e.g. from body tissues or from bacterial cell cultures, various peptide or protein precipitation methods are known in the art.
Especially in the field of plasma proteins for clinical use there exists an industrial fractionation process (the so-called Cohn-process, E. J. Cohn et al, J. Chem. Am. Soc. 68, 1946, 459 and U.S. Pat. No. 2,390,074 which are hereby incorporated by reference) which involves a number of batch processing steps including operations like precipitation and centrifugal separation or filtration of the precipitated proteins. The separation and the yield are improved by a larger and more uniform particle size of the precipitate. In this context, regard is also given to Chan et al. Biotech. Bioeng. XXVIII (1986), 387-393, Chang, Bioeng. 31 (1988), 841-846, Raphael et al. Can. J. Chem. Eng. 73 (1005), 471-483 and U.S. Pat. No. 4,067,863, which are all incorporated by reference.
However, since these properties are generally impaired by super saturation of the precipitant (ethanol EtOH or/and buffer) in consequence of insufficient mixing and excessive particle stress, it has been shown for many applications that conventional batch reactors are not optimal, where the mixing of the precipitant, the precipitation, the growth and the ripening of particles is carried out all in one stirred tank. Another result of insufficient mixing may be additional yield loss due to over heating (heat of dissolution) and harmful pH values.
It is therefore an object of the present invention to provide a method for the precipitation for peptides and/or proteins, which allows for many applications a more controlled precipitation with higher yields.
Such a method is disclosed in claim 1 of the present invention. Accordingly, a method for the precipitation for peptides is disclosed comprising the steps of
The term “spacially separated” especially means and/or includes that the steps a) and b) are carried out in two different vessels (which are connected with each other via pipes etc.); however, the term “spacially separated” also especially means and/or includes that the steps a) and b) may be carried out in two separated parts of one vessel, e.g. a fluid reactor.
The term “precipitation aid” especially includes and/or means any chemical compound or any mixture of chemical compounds which are able to adjuve or to cause the precipitation of peptides out of aqueous solution. According to a preferred embodiment of the present invention, the precipitation aid is selected out of the group of organic solvents comprising ethanol, salt solutions, acids, pH-buffers, phenol, non ionic polymers, ionic polyelectrolytes or mixtures thereof.
The term “peptides” especially includes also substituted and unsubstituted peptides and/or proteins, the substitutions may be—but not limited to—glycosides, nucleic acids, alkyl groups, aryl groups or mixtures thereof, whereby the peptide backbone and/or the side groups may be substituted.
By using this method, within a wide range of applications at least one or more of the following advantages may be reached:
According to an embodiment of the present invention, the macroscopic mixing time in step a) is ≧1 ms and ≦1000 ms, according to an embodiment of the present invention, the macroscopic mixing time in step a) is ≧10 ms and ≦100 ms
According to an embodiment of the present invention, the average mixing velocity (average velocity inside the mixing tube) in step a) is ≧0.05 m/s and ≦5 m/s. This may help within a wide range of applications to keep the time in step a) as little as possible. According to an embodiment of the present invention, the average mixing velocity in step a) is ≧0.2 m/s and ≦1.5 m/s, according to an embodiment of the present invention, the average mixing velocity in step a) is ≧0.3 m/s and ≦1 m/s.
According to an embodiment of the present invention, the pressure drop inside the jet mixer in step a) Δp is ≧0.05 bar and ≦20 bar. This has been shown within a wide range of applications with the present invention to further improve step a). According to an embodiment of the present invention, in step a) Δp is ≧0.1 bar and ≦2.5 bar. according to an embodiment of the present invention, in step a) Δp is ≧0.2 bar and ≦1 bar.
According to an embodiment of the present invention, step a) is performed in a jet mixer with at least two inlets, one for the peptide solution and the precipitation aid, respectively.
In this regard it is especially preferred that the ratio d1 (the diameter of the inlet of the peptide solution) to D (which is the width of the mixing chamber) is ≧0.1:1 and ≦0.4:1, more preferred ≧0.2:1 and ≦0.3:1.
In this regard it is especially preferred that the ratio d2 (the diameter of the inlet of the precipitation aid) to D (which is the width of the mixing chamber) is ≧0.05:1 and ≦0.3:1, more preferred ≧0.08:1 and ≦0.13:1.
According to an embodiment of the present invention step b) is performed while continuously agitating with at least one impeller producing marginal particle stress. According to an embodiment of the present invention step b) is performed while continuously agitating with at least one blade agitator. It has been shown in a wide range of applications within the present invention that this may help to improve the precipitation in step b)
The term “blade agitator” especially means or includes that several radial orientated blades with no inclination to the vertical may be provided on a common axis; if such a blade agitator is used, it is especially preferred that the number of blades is ≧3 and ≦9, preferably ≧4 and ≦6.
According to an embodiment of the present invention, step b) is performed while continuously and/or interrupted agitating or agitating with stepwise changed agitation power with ≧0.01 W/m3 and ≦50 W/m3. Such a measure has shown in practice for a wide range of applications to lead to a good precipitation behaviour of the peptide, especially leading to a bigger average size of a more stable precipitate. According to an embodiment of the present invention, step b) is performed while agitating with ≧0.1 W/m3 and ≦20 W/m3, according to an embodiment of the present invention, step b) is performed while agitating with ≧1.5 W/m3 and ≦5 W/m3
According to an embodiment of the present invention, step b) is performed while agitating with an agitator having no inclined blades and therefore mainly producing radial flow.
According to an embodiment of the present invention, the agitator is set with an extrentricity of ≧0 and ≦0.15. The term excentricity in the context of the present invention especially means and or includes the term e/D with e being the distance from the edge of the agitator to the wall of the vessel and/or the compartment where step b) is performed and D being the diameter of the vessel and/or the compartment where step b) is performed.
Such a measure has shown in practice for a wide range of applications to improve the precipitation as well, since then the mixing behaviour of the agitator may be greatly enhanced.
According to an embodiment of the present invention, the agitator is set with an extrentricity of ≧0.1 and ≦0.05, according to an embodiment of the present invention, the agitator is set with an extrentricity of ≧0.03 and ≦0.015.
According to an embodiment of the present invention, the ratio of the blade diameter d to the diameter D of the vessel and/or the compartment where step b) is performed is d:D≧0.4:1 and ≦0.7:1. By doing so, a continuously improved stirring behaviour with decreasing particle stress of the agitator can be ensured for a wide range of applications within the present invention.
According to an embodiment of the present invention, the ratio of the blade diameter d to the diameter D of the vessel and/or the compartment where step b) is performed is d:D≧0.45:1 and ≦0.65:1, According to an embodiment of the present invention, d:D is ≧0.5:1 and ≦0.6:1
According to an embodiment of the present invention, the blade height to diameter ratio of the at least one blade agitator is h:d≧0.15:1 and ≦1.3:1. with h being the height and d being the diameter. In case the blade agitator comprises several blades it is especially preferred that all of the blades obey this ratio. According to an embodiment of the present invention, the height:width ratio of the blade(s) of the blade agitator is h:d≧0, 25:1 and ≦1:1.
According to an embodiment of the present invention, the ratio of the vessel and/or the compartment where step a) is performed towards the vessel and/or the compartment where step b) is performed is ≧0.01:1 and ≦0.1:1. It has been surprisingly shown that for a wide range of applications it may be advantageous to have a relatively small mixing chamber (i.e. the vessel where step a) is performed) in relation to the precipitation vessel, since then the precipitation aid may be present in a rather great excess during step a).
According to an embodiment of the present invention, the ratio of the vessel and/or the compartment where step a) is performed towards the vessel and/or the compartment where step b) is performed is ≧0.02:1 and ≦0.08:1.
According to an embodiment of the present invention, the method comprises a step a1) between steps a) and b):
This has been shown to be advantageous for a wide range of applications within the present invention since then a deterioration or denaturation of the protein may be avoided.
According to an embodiment of the present invention, the method comprises a step c) after steps a) and b):
By doing so, within a wide range of applications the precipitation step may be run continuously or batch-like which may help to improve the precipitation of the peptide.
According to an embodiment of the present invention, the method comprises a step a2) and a3) between steps a1) and b):
The invention furthermore relates to a device for carrying out the method according to the invention.
The aforementioned components, as well as the claimed components and the components to be used in accordance with the invention in the described embodiments, are not subject to any special exceptions with respect to their size, shape, material selection and technical concept such that the selection criteria known in the pertinent field can be applied without limitations.
Additional details, characteristics and advantages of the object of the invention are disclosed in the subclaims and the following description of the respective figures—which in an exemplary fashion—show several preferred embodiments of devices for performing the method according to the invention.
A second vessel 20, where the peptide to be precipitated (e.g. in an aqueous solution together with other body fluids such as carbohydrates, nucleic acids etc.) is provided, is also in connected to the jet mixer 30 via a second pump 25.
In the jet mixer 30, step a) of the method according to the present invention is carried out; the mixing step is preferably performed as previously described. After the mixing step, at least some of the heat is removed by the heat exchanger 40 and then the mixture is delivered into the precipitation vessel 50.
It should be noted that
It is according to an embodiment of the present invention especially preferred that the tube which delivers the mixture into the vessel ends up in a “trumpet-like” section with an angle of is ≧2° and ≦8°.
In the precipitation vessel, and blade agitator 60 is provided which is preferably as described above.
Although not limited to this, the inventors have in practice so far the present invention in essentially two modes, the batch precipitation mode (“A”) and the recirculation mode (“B”).
In the batch precipitation mode, a vast excess of precipitation aid is added to the protein solution at once, which will lead to point “A” in the diagram. The amount of supersaturation (Δc) will lead to almost immediate precipitation of the protein. Since the mixing step a) is fast in relation to the second step b), this can be used within a wide range of applications to speed up the precipitation process.
In the recirculation mode (“B”), only a small excess of precipitation aid is added. Therefore the smaller Δc will cause only a partial precipitation of the protein (as indicated in the diagram). Again, some more precipitation aid is added, and the precipitation awaited. In the end, the sequel of addition-precipitation-addition-precipitation etc. will ideally occur between the saturation curve c* (straight line) and the dotted line.
This recirculation mode has for some applications the advantage that the precipitated peptide will sometimes be more clean, since co-precipitation can be avoided. The reduced over saturation Δc/c* may lead to an increase of the ratio: particle growth to nucleation velocity and may therefore improve the precipitation result.
In this particular embodiment, the precipitation vessel 50 is connected via a line 70 (see
It is especially preferred that the ratio d1 (the inlet of the peptide solution) to D (which is the width of the mixing chamber) is ≧0.1:1 and ≦0.4:1, more preferred ≧0.2:1 and ≦0.3:1.
It is especially preferred that the ratio d2 (the inlet of the precipitation aid) to D (which is the width of the mixing chamber) is ≧0.05:1 and ≦0.3:1, more preferred ≧0.08:1 and ≦0.13:1.
As can be seen in
After the first mixing of precipitant and protein only the ripening vessel 50 is used for the second and further re-circulations (using only re-circulation line “1” in
In this mode the ripening vessel 50 and the vessel 20, which first contains the protein solution, is alternatively used for the mixture. After the first mixing of precipitant and protein solution the mixture of the second re-circulation is transported in the complete empty protein vessel 20. During the third re-circulation run ripening vessel 50 is used again, and so on (using re-circulation lines “1” and “2” in
Compared to re-circulation mode 1 a smaller distance to the saturation curve can be realized for a wide range of applications within the present invention.
According to a further embodiment (not shown in the figs), lines q1 and/or q2 may be provided, which lead to the vessels 10 and 20 respectively. By doing so also some recirculation may take place as described above.
According to a further embodiment (not shown in the figs), lines q1 and/or q2 may be provided, which lead to the vessels 10 and 20 respectively. By doing so also some recirculation may take place as described above.
However, this device is layed out for a “stepwise” precipitation, as indicated by “B” in
According to a further embodiment (not shown in the figs), lines q1 and/or q2 may be provided, which lead to the vessels 10 and 20 respectively. By doing so also some recirculation may take place as described above.
The present invention will be more understood using the following example, which—in a merely illustrative fashion—shows the precipitation of a protein out of a solution using a device and method according to the present invention.
The precipitation was performed in a device according to
The jet mixer was set up according to
After the jet mixer, an heat exchanger with an heat exchange area of A=0.35 m2 was employed.
The throughput of precipitation aid (acetate buffer and ethanol) was q1=68 l/h, protein solution (plasma solution according to Cohn, see ref. above) q2=756 l/h. The pH-value inside the ripening vessel was 7.12<pH<7.28 with a temperature around −3° C.
The ripening vessel used had a diameter of DR=406 mm equipped with a blade agitator having a blade height:diameter ratio of dh/D=0.5. The blade agitator had six blades, having a ratio of agitator: vessel diameter dR/D=0.55. The relative distance from the blade to the vessel was e/DR=0.025.
The mixture of precipitation aid and protein solution was introduced into the vessel by a lance close to the bottom of the vessel having an conical angle (as described above) of about 5°, so that the incoming power of the stream was less than 0.3 W/m3.
The precipitation led to the following results:
Fibrinogen could be separated from the other proteins in a much higher amount (test results gave about 50%) than in standard procedures. Further proteins, such as IgG, α1-Antitrypsin, Plasminogen and Albumin could only be found in the protein solution, i.e. essentially no co-precipitation took place.
The particular combinations of elements and features in the above detailed embodiments are exemplary only; the interchanging and substitution of these teachings with other teachings in this and the patents/applications incorporated by reference are also expressly contemplated. As those skilled in the art will recognize, variations, modifications, and other implementations of what is described herein can occur to those of ordinary skill in the art without departing from the spirit and the scope of the invention as claimed. Accordingly, the foregoing description is by way of example only and is not intended as limiting. The invention's scope is defined in the following claims and the equivalents thereto. Furthermore, reference signs used in the description and claims do not limit the scope of the invention as claimed.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2007/085580 | 11/27/2007 | WO | 00 | 12/23/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60871514 | Dec 2006 | US |
