APPARATUS FOR THE SYNTHESIS OF OLIGONUCLEOTIDES AND PROCESS FOR THE PREPARATION THEREOF

Abstract

The present invention provides apparatuses and methods for the synthesis of oligonucleotides and related compounds. In particular, the present invention allows to effectively prepare reagents to be fed into an apparatus for the synthesis of such oligomers.

Claims

1-23. (canceled)

24. An apparatus for the automated synthesis of oligonucleotides, comprising: a) a reaction vessel (6) connected via a liquid conduit (9) to a waste container (7); b) a liquid supply unit (19) for delivering liquid reagents to the reaction vessel (6); c) a bypass conduit (11), which allows to direct liquid flow from the liquid supply unit (19) into the waste container (7) without passage through the reaction vessel (6); and d) a control unit (8), wherein the liquid supply unit (19) comprises: b-1) at least one mixing device (5), connected to b-2) at least two liquid supply lines (1, 2), each comprising at least one liquid conduit (l1, l2) with n liquid inlets (i1-n, i2-n), where n is an integer between 1 and 25, and at least one pump (p1, p2).

25. The apparatus of claim 24, wherein at least the inner surfaces of the at least one mixing device (5) and of one of the at least two liquid supply lines (1, 2) are made from an acid-resistant material and/or comprise an acid resistant coating.

26. The apparatus of claim 25, wherein at least the inner surfaces of the at least one mixing device (5) and of one of the at least two liquid supply lines (1, 2) are made from an acid-resistant alloy and/or comprise an acid resistant polymeric coating.

27. The apparatus of claim 24, wherein the liquid supply unit (19) further comprises a third liquid supply line (3) connected to the at least one mixing device (5), the third liquid supply line (3) comprising at least one liquid conduit (13) with n liquid inlets (i3-n), where n is an integer between 1 and 25, and at least one liquid pump (p3).

28. The apparatus of claim 24, wherein the at least one mixing device (5) comprises a static mixer.

29. The apparatus of claim 24, further comprising n sensors (s1 to sn), where n is an integer equal to or larger than 1, which sensor(s) (s1 to sn) is/are positioned downstream of the at least one mixing device (5) and determine(s) at least one property of the liquid emerging from the mixing device (5).

30. The apparatus of claim 29, wherein at least one readout provided by at least one of the sensor(s) (s1 to sn) is used as a feedback signal to regulate the activity of one or more of the pumps comprised in the liquid supply unit (19).

31. The apparatus of claim 29, wherein at least one readout provided by at least one of the sensor(s) (s1 to sn) is used to control whether the liquid flow is directed into the reaction vessel (6) or into the waste container (7) without passage through the reaction vessel (6).

32. The apparatus of claim 24, further comprising at least one flow sensor (24) integrated into each of the at least two liquid supply lines (1, 2) upstream of the at least one mixing device (5), wherein at least one readout provided by the flow sensors is used as a feedback signal to regulate the activity of one or more of the at least one pump (p1, p2).

33. The apparatus of claim 24, wherein the liquid supply unit (19) further comprises at least one heat exchanger (13).

34. The apparatus of claim 29, comprising a conductivity sensor, a temperature sensor, and a UV absorption sensor.

35. The apparatus of claim 24, wherein the reaction vessel (6) is a packed column reactor or a batch reactor.

36. The apparatus of claim 24, further comprising a recycling circuit directing flow from the liquid conduit (9), which connects the reaction vessel to the waste container, back into the reaction vessel (6) by a pump (17).

37. The apparatus of claim 36, wherein the recycling circuit is comprised in one of the at least two liquid supply lines (1, 2), which is connected via one of its inlets (i1-n, i2-n) and a multi way valve (12) to the liquid conduit (9), and whose pump (p1, p2) may drive the recirculation of the fluid from the liquid conduit (9) via the at least one mixing device (5) back into the reaction vessel.

38. A method of assembling an oligonucleotide chain in automated fashion by repeated cycles of building block coupling, wherein the following steps 1 through 6 are carried out anew in each of the cycles of building block coupling: 1. providing a n-mer oligonucleotide, where n is an integer equal to or larger than 1, bound to a solid support and comprising a first reactive group free to extend the oligonucleotide backbone by reaction with a second reactive group comprised in a building block to be incorporated; 2. providing the building block to be incorporated, which comprises the second reactive group free to react with the first reactive group of the n-mer oligonucleotide, and which further comprises a first reactive group blocked by an acid sensitive temporary protecting group; 3. contacting the n-mer oligonucleotide with the building block to be incorporated under conditions that allow for binding of the first reactive group of n-mer oligonucleotide to the second reactive group of the building block to be incorporated, and generating a protected, extended n-mer oligonucleotide, which is blocked from further extension by the acid sensitive temporary protecting group; 4. preparing an acidic deprotection reagent by mixing at least two liquid compositions; 5. contacting the protected, extended n-mer oligonucleotide of step 3 with the acidic deprotection reagent of step 4, thereby cleaving the acid sensitive temporary deprotecting group from the extended n-mer oligonucleotide; and 6. removing the deprotection reagent and soluble cleavage product from the extended n-mer oligonucleotide, which may then be used as the n-mer oligonucleotide in step 1 of the following coupling cycle.

39. The method of claim 38, wherein the composition of deprotection reagent is different between at least two coupling cycles.

40. The method of claim 38, wherein the composition of the deprotection reagent is defined individually for each coupling cycle.

41. The method of claim 38, wherein the content of acid in the deprotection reagent is selected from the range of 0.1% (w/w) to 50% (w/w) acid individually for each coupling cycle.

42. The method of claim 38, wherein the acid sensitive temporary protecting group is a trityl type protecting group.

43. The method of claim 38, wherein the first reactive group is a hydroxyl group.

44. The method of claim 43, wherein the second reactive group is selected from the group consisting of a phosphoamidite group and a H-phosphonate monoester group.

45. The method of claim 44, wherein step 3 comprises contacting the building block and the n-mer oligonucleotide under conditions, which allow for formation of a phosphite triester group, and mixing the resulting compound with an oxidizing reagent or a sulfurizing reagent to convert the phosphite triester group into a phosphate triester group or a thiophosphate triester group, respectively.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0116] The figures show

[0117] FIG. 1 a basic embodiment of an apparatus for the automated synthesis of oligomers having four liquid supply lines,

[0118] FIG. 2 a further embodiment of the apparatus additionally comprising a recycling conduit,

[0119] FIG. 3 a further embodiment of the apparatus where one of the liquid supply lines serves as recycling circuit,

[0120] FIG. 4 a further embodiment of the apparatus having storage vessels,

[0121] FIG. 5 another embodiment of the apparatus comprising a manifold,

[0122] FIG. 6 another embodiment of the apparatus comprising two mixing devices and a manifold,

[0123] FIG. 7 a further embodiment of the apparatus having two liquid supply lines,

[0124] FIG. 8 another embodiment of the apparatus having two liquid supply lines and a recycling circuit,

[0125] FIG. 9 an embodiment of the apparatus of FIG. 7 with additional sensors arranged downstream of the mixing device,

[0126] FIG. 10 an embodiment of the apparatus of FIG. 3 with only two liquid supply lines,

[0127] FIG. 11 an embodiment of the apparatus of FIG. 2 with only three liquid supply lines,

[0128] FIG. 12 another embodiment of the apparatus having three liquid supply lines, wherein one of the liquid supply lines serves as recycling circuit, and

[0129] FIG. 13 a further embodiment of the apparatus of FIG. 3 with only three liquid supply lines.

[0130]

TABLE-US-00004 List of reference signs  1 liquid supply line 1 i1-1 inlet no. 1 of line 1 i1-2 inlet no. 2 of line 1 i1-3 inlet no. 3 of line 1 i1-4 inlet no. 4 of line 1 i1-n inlet no. n of line 1 p1 liquid pump of line 1 I1 liquid conduit of line 1  2 liquid supply line 2 i2-1 inlet no. 1 of line 2 i2-2 inlet no. 2 of line 2 i2-3 inlet no. 3 of line 2 i2-4 inlet no. 4 of line 2 i2-n inlet no. n of line 2 p2 liquid pump of line 2 I2 liquid conduit of line 2  3 liquid supply line 3 i3-1 inlet no. 1 of line 3 i3-2 inlet no. 2 of line 3 i3-3 inlet no. 3 of line 3 i3-4 inlet no. 4 of line 3 i3-n inlet no. n of line 3 p3 liquid pump of line 3 I3 liquid conduit of line 3  4 liquid supply line 4 i4-1 inlet no. 1 of line 4 i4-2 inlet no. 2 of line 4 i4-3 inlet no. 3 of line 4 i4-4 inlet no. 4 of line 4 i4-n inlet no. n of line 4 p4 liquid pump of line 4 I4 liquid conduit of line 4 v1-n storage vessel no n of line 1 v2-n storage vessel no n of line 2 v3-n storage vessel no n of line 3 v4-n storage vessel no n of line 4  5 mixing device  6 reaction vessel  7 waste container  8 control unit  9 controllable liquid conduit 10 valve 11 bypass conduit s1 sensor 1 s2 sensor 2 sn sensor n 12 3 way valve 13 heat exchanger 14 manifold 15 local control unit 16 liquid recycling conduit 17 liquid pump 18 liquid inlet 19 liquid supply unit 20 liquid conduit between mixing device and reaction vessel 21 liquid conduit between mixing device and manifold 22 liquid conduit between manifold and reaction vessel 23 sensor 24 flow sensor

DESCRIPTION OF THE FIGURES

[0131] FIG. 1 shows a basic embodiment of the apparatus according to the present invention. The liquid supply unit 19 of the apparatus may comprise a mixing device 5, which is connected to four liquid supply lines, a first liquid supply line 1, a second liquid supply line 2, a third liquid supply line 3 and a fourth liquid supply line 4. Each of said liquid supply lines comprises a pump [(p1), (p2), (p3), or (p4), respectively], a liquid conduit [(l1), (l2), (l3), or (l4), respectively], and n inlets [(i1-1 to i1-n), (i2-1 to i2-n), (i3-1 to i3-n), (i4-1 to i4-n), respectively], where n is an integer equal to or greater than 1. The fluid connection between each of the liquid inlets and the conduit may be regulated by means of a valve 10. The liquid supply lines 1, 2, 3, 4 are highlighted by dashed boxes for the sake of clarity. It is understood that the entire length of the liquid conduits [(l1), (l2), (l3), or (l4), respectively] up to the inlet of the mixing device 5 is considered as belonging to the respective liquid supply line. The mixing device 5 unites any flows from said liquid supply lines 1, 2, 3, 4 and mixes them so as to obtain one homogenous solution, which may be led though a liquid conduit 20 comprising n sensors (s1 to sn), where n is an integer equal to or greater than 1, into the reaction vessel 6. The sensors may provide a feedback signal correlating with the composition of the solution to a control unit 8, which signal is used by the control unit 8 to regulate the flow of the line's pumps [(p1), (p2), (p3), or (p4), respectively]. As long as the composition of the solution passing the sensors is not stable within a given set interval, the solution will not be directed into the reaction vessel, but will be guided via an automated three way valve 12 and a bypass conduit 11 into a waste container 7. Further to the pumps p1, p2, p3, p4, the control unit 8 controls the liquid flow through the conduits 11, 20, and through the liquid conduit 9 connecting the reaction vessel with the waste container 7. The control unit 8 may thereby determine which solution is incubated for which duration of time with the contents of the reaction vessel.

[0132] FIG. 2 shows a further embodiment of the inventive apparatus, to which all of the remarks re. FIG. 1 apply. In addition to the above-discussed elements, a liquid recycling conduit 16 with a pump 17 is connected to the liquid conduit 9 via a three way valve 12. This setup allows to circulate a given solution coming out of the reaction vessel 6 via the mixing device 5 and the liquid conduit 20 with the n sensors (s1 to sn) back into the reaction vessel 6. The actions of the three-way valve 12 and of the pump 17 are controlled by the control unit 8. Additional liquid inlets 18 on the recycling conduit 16 allow for flexibility.

[0133] FIG. 3 shows a further embodiment of the inventive apparatus, to which all of the remarks re. FIG. 1 apply. In addition to the above-discussed elements, a heat exchanger 13 is integrated into the liquid conduit 20 between the mixing device 5 and the first sensor s1. This allows to regulate the temperature of the solution in order to obtain more reliable readouts from the sensors and to regulate the temperature of the reagents within the reaction vessel 6. Moreover, a further sensor 23 is integrated into the liquid conduit 9 downstream from the reaction vessel. This sensor likewise provides a signal to the control unit, which may correlate with events inside the reaction vessel. The second liquid supply line 2 in this embodiment serves a double function in that one of its inlets is connected to the liquid conduit 9 via a three way valve 12 positioned between the sensor 23 and the waste container 7. This setup allows to circulate a given solution coming out of the reaction vessel 6 via the liquid conduit 12, the mixing device 5 and the liquid conduit 20 with the n sensors (s1 to sn) back into the reaction vessel 6, but without the need for a dedicated recycling conduit and pump as shown in FIG. 2. The actions of the three-way valve 12 and of the pump p2 are controlled by the control unit 8.

[0134] FIG. 4 shows a further embodiment of the inventive apparatus, to which all of the remarks re. FIG. 3 apply. Three liquid supply lines 1, 3, 4 are shown with four storage vessels each [(v1-1 to v1-4), (v3-1 to v3-4), (v4-1 to v4-4), respectively] and n−4 inlets [(i1-5 to i1-n), (i3-5 to i3-n), (i4-5 to i4-n), respectively], where n is an integer equal to or greater than 7. The second liquid supply line 2 serves a double function of liquid supply line and recycling conduit as explained with reference to FIG. 3 above. It is shown with two storage vessels (v2-1), (v2-2) and n−2 inlets (i2-3 to i2-n), where n is an integer equal to or greater than 5. The liquid supply unit 19 comprises a heat exchanger 13 and two sensors (s1), (s2).

[0135] FIG. 5 shows a further embodiment of the inventive apparatus. The liquid supply unit 19 of the apparatus may comprise a mixing device 5, which is connected to four liquid supply lines 1, 2, 3, 4. Each of said liquid supply lines comprises a pump [(p1), (p2), (p3), or (p4), respectively], a liquid conduit [(l1), (l2), (l3), or (l4), respectively], and n inlets [(i1-1 to i1-n), (i2-1 to i2-n), (i3-1 to i3-n), (i4-1 to i4-n), respectively], where n is an integer equal to or greater than 1. The fluid connection between each of the liquid inlets and the conduit may be regulated by means of a valve 10. The combined solution emerging from the mixing device 5 is directed via a liquid conduit 21 into a manifold 14. Integrated into said liquid conduit 21 are a heat exchanger 13 and a sensor (s1). The sensor (s1) provides signals to a local control unit 15, which regulates the action of the liquid supply line's pumps (p1), (p2), (p3), or (p4). The local control unit 15 may be under the control of the control unit 8, or it may be independent. The manifold 14 is further connected to four additional liquid conduits, each comprising a pump 17 and two liquid inlets 18. The liquid flow emerging from the manifold is directed through a liquid conduit 22 into the reaction vessel 6. An additional heat exchanger 13 and a sensor 23 are integrated into said liquid conduit 22. All other elements are as set out with respect to FIG. 3.

[0136] FIG. 6 shows a further embodiment of the inventive apparatus, which is similar to that of FIG. 5. The manifold 14 receives input from two mixing devices 5, each connected to four liquid supply lines 1, 2, 3, 4.

[0137] FIG. 7 shows a further embodiment of the apparatus in which the apparatus has two liquid supply lines. The apparatus of FIG. 7 essentially corresponds to the apparatus described with respect to FIG. 1, but comprises only two liquid supply lines 1, 2 and does not include sensors arranged downstream of the mixing device 5.

[0138] FIG. 8 shows another embodiment of the apparatus having two liquid supply lines and a recycling circuit. The apparatus of FIG. 8 essentially corresponds to the apparatus as described with respect to FIG. 7, but additionally includes the recycling circuit comprising the liquid recycling conduit 16 and the liquid pump 17. The recycling circuit as shown in FIG. 8 also comprises additional liquid inlets 18.

[0139] Further, the embodiment of FIG. 1 comprises liquid flow sensors 24 arranged in each of the two liquid supply lines 1, 2 which are arranged downstream of the respective liquid pump p1, p2 and upstream of the mixing device 5.

[0140] FIG. 9 shows an embodiment of the apparatus of FIG. 1 with only two liquid supply lines 1, 2. The explanations provided with respect to FIG. 1 also apply to the embodiment shown in FIG. 9, except for the omission of the two further liquid lines 3, 4.

[0141] FIG. 10 shows an embodiment of the apparatus as described with respect to FIG. 3 but having only two liquid supply lines.

[0142] FIG. 11 shows an embodiment of the apparatus of FIG. 2 with only three liquid supply lines. Except for the reduction of the number of liquid supply lines 1, 2, the description of the embodiment of FIG. 2 also applies to the embodiment of FIG. 11.

[0143] FIG. 12 shows another embodiment of the apparatus having three liquid supply lines, wherein one of the liquid supply lines serves as recycling circuit. The embodiment of FIG. 12 is similar to the embodiment described with respect to FIG. 3, except for the omission of the sensors and the fourth liquid supply line.

[0144] FIG. 13 shows a further embodiment of the apparatus of FIG. 3 with only three liquid supply lines. In contrast to the embodiment of FIG. 12, the sensors s1 . . . sn as well as sensor 23 are present.