Peptide synthesizer with series resin reactors

Abstract

A solid phase peptide synthesis (SPPS) apparatus and method of using it to manufacture peptides is taught herein. The system comprises at least two reactors, each reactor comprising an amount of SPPS resin. The reactors are disposed in series. A deprotection reagent is added to the first reactor, then sequentially transferred to the second and third reactors, thereby for deprotecting the protected N-group. A washing solvent is added to the first reactor, then transferred to the second reactor, and this operation is repeated several times. Likewise, an amino acid activated ester solution is sequentially added to the first, second and third reactors, thereby for coupling the amino acid to the deprotected N-group. A washing solvent is added to the first reactor, then transferred to the second reactor, and this operation is repeated several times before the next cycle. The use of reactors in series reduces the total solvent required. On-line LCMS is also used to monitor the progress and nature of the reactions taking place within the solid phase resin particles.

Description

Technical Field

[0001] This disclosure relates to a novel system and method for synthesizing peptides. More specifically, this disclosure relates to an apparatus that uses tandem resin reactors as part of a solid-phase peptide synthesis mechanism for coupling peptides together.

[0002] background

[0003] Solid-phase peptide synthesis (“SPPS”) is the most commonly used method and system for synthesizing peptides and amino acid sequences. SPPS involves coupling an activated amino acid (which is typically the terminal or last amino acid in a sequence) to a solid support. This solid support is typically a polymer resin bead functionalized (e.g., with an NH2 group). The terminal amino acid (usually with its NH2 terminus protected by an F-moc, BOC, or other protecting group) reacts with the resin to allow the functionalized groups on the resin to react and bind with the activated COOH group of the terminal amino acid. In this way, the terminal amino acid is covalently linked to the resin.

[0004] Then, in the next step, the NH2 terminus of the terminal amino acid is deprotected, thereby exposing its NH2 group for the next reaction. Accordingly, a new amino acid is introduced. The NH2 terminus of this new amino acid is protected by a protecting group (such as F-moc, BOC, or another protecting group). Therefore, when this new amino acid is added, the activated ester from this new amino acid reacts with the newly deprotected NH2 group of the terminal amino acid, thereby coupling the two amino acids together. Once this new amino acid has been coupled, it also has a protected NH2 group, which can subsequently be deprotected and reacted with the next amino acid. By repeatedly performing this iterative process, the entire amino acid sequence can be constructed. Once the entire sequence has been constructed, it can be uncoupled (cleaved) from the resin and deprotected, thereby yielding the amino acid structure. (It should be noted that the side chains of various amino acids (R1, R2, etc.) added in this way can be orthogonally protected by groups such as BOC, tert-butyl, or triphenylmethyl to prevent these side chains from reacting during amino acid synthesis. One or more amino acids may also have "side chains" or other groups as part of their structure, which may also need to be protected. However, those skilled in the art will recognize how such side chains or other groups are constructed, protected, and subsequently deprotected during synthesis.)

[0005] Although the SPPS method is commercially available and remains standard in peptide synthesis, its drawbacks include its high cost and time consumption. Each added amino acid must be deprotected and coupled, which is difficult and often results in the use of large amounts of solvent. Worse still, many of these solvents are not environmentally friendly.

[0006] Therefore, finding new ways to use SPPS to address one or more of these shortcomings would be an improvement, especially in the commercial manufacture of peptides. Further progress would be made if such a system were more environmentally friendly and reduced manufacturing costs. In fact, this embodiment explicitly reduces waste as well as the amount of solvents and reagents used. Such a method and system are disclosed herein.

[0007] Overview

[0008] A method and system for coupling an activated ester of amino acid “X” to a protected N-group (such as an NH2 terminus) of an amino acid attached to an SPPS resin. Typically, the system comprises a series of reactors arranged in series. In some embodiments, two or more reactors are arranged in series. In a preferred embodiment, three or more reactors are arranged in series.

[0009] Each reactor contains a certain amount of protected N-groups attached to the peptide synthesis resin. These protected N-groups can be NH2 groups of amino acids or NH2 groups present or covalently attached to the resin itself, such as, but not limited to, Sieber amide or Rink Amide resins. Other types of resins, such as Wang resin or CTC (triphenylchloromethyl chloride) resin, can also be used.

[0010] The first step of this method involves adding a first amount of deprotecting agent to a first reactor and allowing this agent to contact the protected N-group. Then, the first amount of deprotecting agent is transferred from the first reactor to a second reactor, and a second amount of deprotecting agent is transferred to the first reactor. The first amount of deprotecting agent is removed from the second reactor, and the second amount of deprotecting agent is transferred from the first reactor to the second reactor. The second amount of deprotecting agent is then removed from the second reactor.

[0011] The purpose of contacting the first and second reactors with the first and second amounts of deprotecting agents is that these agents react with the protected N-groups and, individually or jointly, function in both reactors to deprotect the protected N-groups attached to the peptide synthesis resin. Thus, by adding the first and second amounts of deprotecting agents, the N-groups in both reactors are deprotected and can be coupled to another amino acid. A first amount of wash solution is added to the first reactor, then transferred to the second reactor, and then to waste. The washing cycle is repeated several times. Washing with solvents can use green solvents commonly used in SPPS or more environmentally friendly solvents. Such green washing solvents include acetonitrile (ACN), ethyl acetate, isopropyl acetate, 2-MetHF (2-methyltetrahydrofuran), and CPME (cyclopentylmethyl ether), or solvent mixtures such as NBP / THF 2 / 1 v / v, as exemplified here. These chemical reactions can also be carried out in ACN, ACN / DMSO, or n-butylpyrrolidone, which are also green solvents.

[0012] Accordingly, a first amount of amino acid "X" and a first amount of solvent are added to a first reactor, and then, after a certain period of time, the mixture is transferred from the first reactor to a second reactor. It should be noted that the amount of amino acid "X" added to the reactor is actually an "activated ester" of amino acid X, thereby promoting the coupling reaction. However, for the sake of brevity, it may be simply referred to herein as adding "a certain amount of amino acid X" to the reactor, but those skilled in the art will recognize that it is an activated ester. Alternatively, unactivated amino acids may be added to the reactor, followed by the addition of an activation solution to cause the amino acids to react and convert into activated esters. As used herein, this also falls within the meaning of "adding an activated ester of amino acid to the resin."

[0013] A second amount of amino acid "X" and a second amount of solvent are added to the first reactor. The first and second amounts of amino acid "X" activate the ester, individually or jointly, to couple amino acid "X" to the deprotected N group in the first reactor (the activated ester of amino acid "X" can be any amino acid, including functionalized, derivatized, or synthetic amino acids that are desired to be added to the chain). (As used herein, the coupling of amino acid "X" is sometimes mentioned, but those skilled in the art will recognize that, for ease of reaction, the activated ester is most commonly used).

[0014] A first amount of amino acid "X" and a first amount of solvent are removed from the second reactor, and a second amount of amino acid "X" and a second amount of solvent are transferred from the first reactor to the second reactor. The second amount of amino acid "X" and the second amount of solvent are then removed from the second reactor. The first and second amounts of amino acid "X," individually or jointly, couple amino acid "X" to the deprotected N group in the second reactor. A first amount of solvent wash is added to the first reactor, then transferred to the second reactor, and then to waste. The solvent washing cycle is repeated several times. While the first amount of solvent wash is in the second reactor, the second amount of solvent wash may be in the first reactor, and so on. Therefore, after these steps, amino acid "X" is coupled to the deprotected N group, thereby adding amino acid "X" to the chain. Of course, as with other SPPS systems, amino acid "X" contains a protected NH2 group, so the above process can be repeated (e.g., deprotecting the NH2 group and coupling the new amino acid to it in the manner described above). Therefore, by repeating this process, the desired amino acid sequence and / or peptide can be constructed. Once synthesis is complete, the constructed amino acids can be released (uncoupled) into the resin in both the first and second reactors.

[0015] Although the above method uses two reactors in series—each with its own resin supply—other embodiments can be designed in which a third reactor, also containing a certain amount of resin, is connected in series with the first two reactors. In this embodiment, a deprotection step must also be performed in the third reactor. Therefore, once the first amount of deprotecting agent has been removed from the second reactor, it is added to the third reactor. This first amount of deprotecting agent is then removed from the third reactor, and then the second amount of deprotecting agent—once removed from the second reactor—is added to the third reactor. Similarly, the third amount of deprotecting agent is added to the first reactor, moved to the second reactor, and then moved to the third reactor. The purpose of the first, second, and third amounts of deprotecting agent is to deprotect the protected N-groups attached to the peptide synthesis resin in the third reactor, individually or collectively. In a similar manner, the first amount of amino acid "X" and the first amount of solvent to the third reactor are added to the third reactor after being removed from the second reactor. Similarly, the second amount of amino acid "X" and the second amount of solvent to the third reactor are added to the third reactor after being removed from the second reactor. The third amount of amino acid "X" and the third amount of solvent are sequentially recycled through the first, second, and third reactors. The first, second, and third amounts of amino acid "X" are coupled individually or collectively to the deprotected N group in the third reactor. Then, the washing step described above is performed. Thus, in this way, amino acid sequences can be iteratively constructed in all three reactors (by repeating these or similar steps for each amino acid), and then released from the resin in each of the three reactors.

[0016] Therefore, this embodiment provides various reactors arranged in series, with reagents added to a first reactor, then sequentially moved to a second reactor, then a third reactor, and so on. By arranging these reactors in series, each reactor can contain a certain amount of resin for SPPS, which is used to construct peptide sequences. However, by arranging the reactors in this way, a smaller amount of solvent (washing material) is required. Similarly, a smaller amount of coupling reagent may be needed, resulting in a less wasteful and more efficient and environmentally friendly method. Brief description of the attached diagram

[0018] The features and advantages of this disclosure will become apparent to those skilled in the art when considered in conjunction with the following detailed description in conjunction with the accompanying drawings.

[0019] Figure 1 This is a schematic view of the system and method for coupling amino acid “X” to a protected N-group attached to the peptide synthesis resin used herein;

[0020] Figure 2 This is a schematic view of the system and method for coupling amino acid “X” to a protected N-group attached to the peptide synthesis resin used herein;

[0021] Figure 3 This is a schematic view of the system and method for coupling amino acid “X” to a protected N-group attached to the peptide synthesis resin used herein;

[0022] Figure 4 Is with Figure 3 A diagrammatic view of the systems and methods used in the online LCMS.

[0023] Figure 5 This is a perspective view of three reactors connected in series as used in this embodiment;

[0024] Figure 6 Shows peptides that can be prepared using this embodiment; and

[0025] Figure 7 This demonstrates peptides that can be prepared using this implementation scheme.

[0026] Detailed Explanation

[0027] To facilitate understanding of the principles of this disclosure, reference will now be made to the embodiments shown in the accompanying drawings, and specific terminology will be used to describe them. Nevertheless, it should be understood that this is not intended to limit the scope of the invention.

[0028] Now for reference Figure 1A schematic view of system 100 for coupling amino acid "X" to a protected N-group attached to a peptide synthesis resin. System 100 implements the methods outlined herein and is a modified SPPS system designed for the production of peptide and / or amino acid sequences. System 100 comprises at least two reactors, depicted as a first reactor 106 and a second reactor 108. These reactors 106, 108 are arranged in series. More than two reactors can be used. In fact, in Figure 1 In the system 100 shown, a third reactor 112 is also arranged in series. More than three reactors may also be used.

[0029] Each reactor contains a certain amount of resin 116. The resin includes a protected N-group 120 (such as a protected NH2 group). In some embodiments, this protecting group for the protected N-group is an Fmoc group. Those skilled in the art of SPPS will recognize the types of resins that can be used as resin 116, including Seiber and Rink amide resins. As part of the SPPS process, the protected N-group must be "deprotected" to make it receptive to amino acids (and thus for constructing peptide / amino acid sequences). Therefore, a deprotection process is performed. This deprotection is carried out by adding a first amount of deprotecting agent 126. (This first amount of deprotecting agent 126 is graphically represented by arrows). In some embodiments, the deprotecting agent may be piperidine, but other materials / reagents may also be used.

[0030] A first amount of deprotecting agent 126 can be stirred and allowed to react with the protected N-groups 120 on the resin 116 in the first reactor 106 for a period of time. (Those skilled in the art will appreciate how to determine the exact time quotient required here). The first amount of deprotecting agent 126 is then removed from the first reactor 106 and transferred to the second reactor 108 (as indicated by arrow 128). This first amount of deprotecting agent 126 can be stirred and allowed to react with the protected N-groups 120 on the resin 116 in the second reactor 108 for a period of time. Simultaneously, a second amount of deprotecting agent 130 is added to the first reactor 106 and allowed to react in a similar manner.

[0031] Once these reactions have completed (or in other words, the allotted time has elapsed), the first amount of deprotecting agent 126 is removed from the second reactor 108 and transferred to the third reactor 112 (as indicated by arrow 136). Similarly, the second amount of deprotecting agent 130 is removed from the first reactor 106 and transferred to the second reactor 108 (as indicated by arrow 140). Then, the third amount of deprotecting agent 134 is added to the first reactor 106. At this point, the reactions in the first reactor 106, the second reactor 108, and the third reactor 112 are allowed to continue.

[0032] After the reactions in the first reactor 106, the second reactor 108, and the third reactor 112 have completed (or in other words, the allotted time has elapsed), the first amount of deprotecting agent 126 is removed from the third reactor 112. The second amount of deprotecting agent 130 is removed from the second reactor 108 and transferred to the third reactor 112 (as indicated by arrow 144). The third amount of deprotecting agent 134 can be removed from the first reactor 106 and added to the second reactor 108 (as indicated by arrow 146).

[0033] In some embodiments, the first amount of deprotecting agent 126 removed from the third reactor 112 may be sent to another reactor (if the embodiment includes an additional reactor). In other embodiments, the first amount of deprotecting agent 126 is collected and sent to waste. The same applies to the second and third amounts of deprotecting agents 130, 134 that have been recycled through the third reactor 112. In other embodiments, including Figure 1 In the embodiment shown, this first amount of deprotecting agent 126 (and subsequently the second and third deprotecting agents 130, 134) can be fed back to the first reactor 106 (as indicated by arrow 148) for further iterations of deprotection as needed. (Note that if the deprotecting agent is fed back to the first reactor, this method is more likely to be a batch reaction and may be less efficient.)

[0034] A third amount of reagent 134 is removed from the first reactor 106 and transferred to the second reactor 108. This amount of reagent 134 is then circulated through the second reactor 108 and the third reactor 112 in the manner described above (although the arrow indicating that the third amount of reagent 134 is sent from the second reactor 108 to the third reactor 112 is not shown).

[0035] Those skilled in the art will recognize that the first, second, or third amounts of deprotecting reagent 126, 130, 134 can be pumped to adjust as needed to ensure that the reagent volume in each amount (and in each reactor) is consistent or nearly consistent.

[0036] As will be apparent to those skilled in the art, the deprotecting agents 126, 130, and 134 added to the first, second, and third reactors 106, 108, and 112 are intended, individually or collectively, to deprotect the protected N-group 120 attached to the peptide synthesis resin 116. By tandemly deprotecting the N-group using amounts of reagents cascaded in a manner outlined herein, the N-group is prepared for coupling reactions to couple the N-group to another amino acid (as described in more detail below).

[0037] If necessary, once unprotected, the N-groups attached to resin 116 can be “washed” with a solvent such as DMF. Other solvents may also be used, including NBP (N-butylpyrrolidone), NMP (N-methyl-2-pyrrolidone), DMSO, acetates and ethers such as MeTHF (methyltetrahydrofuran), or solvent mixtures such as NBP / THF 2 / 1 v / v, as illustrated here. Such washing can be performed in the same manner as outlined above for deprotecting agents 126, 130, and 134. In other words, a first amount of washing solvent can be added to the first reactor 106, followed by circulation through the second and third reactors 108 and 112. Similarly, second and third amounts of washing solvent can also be circulated through reactors 106, 108, and 112. This washing step can be iterative, so that there may be 5, 8, or 10 different washing cycles (each time adding the same amount of solvent or a new amount of solvent to the first reactor). (Although not described in detail herein, the washing step may be important and may be performed after each deprotection and coupling step). Similarly, pump calibration can also be used to ensure that the amount of washing solvent added to the reactor is almost consistent each time.

[0038] Now for reference Figure 2 The coupling of amino acids is now described using system 100. Each resin 116 has been deprotected (and optionally washed) as outlined above. Accordingly, resin 116 is shown as comprising unprotected N-groups 120a (rather than) Figure 1 The protected N-group 120 shown.

[0039] The first amount of amino acid "X" 150 is added to the first reactor 106. This is in Figure 2 Arrows are used to represent this graphically. Figure 2 In the illustrated embodiment, a first amount of amino acid “X” 150 has been premixed with a first amount of solvent 152 and a first amount of other reagents 154. More specifically, the first amount of amino acid “X” 150 has been mixed with other reagents 154 and solvent 152 before it is added to the first reactor 106. In other embodiments, the first solvent 152 and / or other reagents 154 may also be added sequentially and / or simultaneously to the first reactor 106 in addition to the first amount of amino acid “X” 150. In some embodiments, the first amount of solvent 152 may be DMF. In some embodiments, the other reagents 154 are reagents for “activating” amino acid “X” 150 and / or unprotected N-group 120a and / or promoting coupling reactions. Thus, in some embodiments, the other reagents 154 may be DIC and oxyma.

[0040] Once amino acid “X” 150 (and a first amount of solvent 152 and other reagents 154) is added to the first reactor 106, the coupling reaction is allowed to proceed. This reaction occurs between the unprotected N-group 120a and amino acid “X”. After a period of time (e.g., 30 minutes or some other set time amount that those skilled in the art understand how to calculate / determine), the first amount of amino acid “X” 150 can be removed from the first reactor 106 (as indicated by arrow 160). In some embodiments, this first amount of amino acid “X” 150 can be transferred to a second reactor 108. The first amount of solvent 152 and / or other reagents 154 can also be removed from the first reactor 106 and transferred to the second reactor 108.

[0041] Once in the second reactor 108, a first amount of amino acid "X" (along with a first solvent 152 and / or other reagent 154) can be used to react with the unprotected N-group 120a in the second reactor 108. Similarly, a second amount of amino acid "X" 170 can be added to the first reactor 106. (This second amount of amino acid "X" 170 can still be premixed with the other reagent 154 and / or solvent 152). After a period of time (e.g., 30 minutes), the first amount of amino acid "X" 150 can be removed from the second reactor 108 (as indicated by arrow 164). This first amount of amino acid "X" 150 can be transferred from the second reactor 108 to the third reactor 112. If the first amount of solvent 152 and / or other reagent 154 is used, they will also be removed from the second reactor 108 and transferred to the third reactor 112. The second amount of amino acid "X" 170 can be removed from the first reactor 106 (as indicated by arrow 174). This second amount of amino acid “X” 170 can be transferred from the first reactor 106 to the second reactor 108.

[0042] A third amount of amino acid "X" 180 can be added to the first reactor 106. (This third amount of amino acid "X" 180 can still be premixed with other reagents 154 and / or solvent 152 and can come from the same batch as the first amount 150 and / or the second amount 170). The reaction is allowed to continue so that the unprotected N-group 120a in the first, second, and third reactors 106, 108, 112 reacts with and couples to amino acid "X". Once this reaction is complete or after a period of time, the first amount of amino acid "X" can be removed from the third reactor 112 and sent to waste or recycled back to the first reactor 106 (as indicated by arrow 188). (Such recycling makes this reaction more like a batch reaction, and is therefore less desirable. In fact, if a batch reaction mode is required, it is better to couple the reactors in parallel). In a similar manner, a second amount of amino acid "X" 170 can be transferred to a third reactor 112 (and react as indicated by arrow 166), and a first amount of amino acid "X" 180 can be transferred to a second reactor 108 and subsequently to a third reactor 112 (and allowed to react in each reactor). In this iterative manner, the reaction proceeds "in series," and the first, second, and third amounts of amino acid "X" 150, 170, and 180 are recycled through reactors 106, 108, and 112. In this way, amounts of amino acids 150, 170, and 180, individually or collectively, are used to react with unprotected N-groups 120a in the first, second, and third reactors 106, 108, and 112 and to couple amino acid "X" to the deprotected N-groups 120a.

[0043] In some embodiments, it may be advantageous to filter the solution before transferring it from one reactor to the next (e.g., from the first reactor 106 to the second reactor 108 or from the second reactor 108 to the third reactor 112). Those skilled in the art will appreciate how such filtration can be performed.

[0044] It is important to recognize that using tandem reactors as outlined herein can reduce the amount of solvent used for deprotection and / or washing steps. For example, if a conventional batch process is used, the deprotection reaction requires a 20% solution of piperidine in DMF. This solution is divided into 10 volumes, and each batch reactor reacts with this solution 3 times. This results in approximately 30 L / kg being used per reactor. However, if three reactors are used in tandem as taught herein, the 20% solution of piperidine in DMF is still divided into 10 volumes based on the amount of resin in each reactor and reacted 3 times (iterations) in the three reactors in the series. To ensure that the number of piperidine equivalents experienced by reactors 2 and 3 is greater than or equal to the batch, a fourth charge of 10 L / kg can be used. Here, the total amount of piperidine solution is 40 / 3 = 13.3 L / kg, a reduction of 2.25 times.

[0045] The higher the number of excess amino acids and the higher the cost of the amino acids, the more advantageous it is to use a series reactor for coupling. For example, the price of Lys20 IV uncoupling reagent may be around $20 / gram; therefore, reducing the amount of excess reagent can significantly save costs. However, due to the retention of the reaction solution in the resin, the lower the number of excess equivalents, the more advantageous it is to run the coupling reaction in parallel to prevent a certain percentage of excess equivalents from being transferred to the next reactor without dilution. If the excess amino acid equivalents are about 2 or less and if they are standard, inexpensive amino acids, then running the coupling reaction in parallel but running it in series for deprotection and washing is preferable.

[0046] It should be noted that in some implementations, the greater the number of reactors in series, the less total solvent and reagents are required per kilogram of product. This is analogous to the principle of flow chemistry, where a larger number of CSTRs (continuous stirred tank reactors) in series brings the flow closer to an ideal plug flow. In some implementations, it is believed that three reactors in series may be optimal due to the trade-off between waste reduction and equipment costs. In other words, implementations can be designed where diminishing returns are achieved by connecting more than three to five reactors in series. To achieve the same maximum residual reagent concentration at the end of washing, three reactors in series can reduce solvent requirements by half compared to a single batch. Of course, other implementations can be designed where more than three or five reactors are used. Other implementations can be designed where two reactors are used in series.

[0047] In some implementations, it may be necessary to measure all reagent charges by mass and use the DeltaV distributed control system (provided by Emerson, St. Louis Missouri USA) instead of a Windows-based user interface. This is because archiving is also automated in DeltaV, and the system creates a master batch record of executed operations. Furthermore, in a DeltaV system, most operations are performed remotely, which can be done from anywhere in the world with internet access. DeltaV systems can be set up to send descriptive text messages to operators, chemists, engineers, and analysts' mobile phones when important steps occur in the process or when anything requires attention, and then handle the situation remotely in most cases. One of the common problems with the best commercially available lab-scale SPPS technologies is a control system crash, missed charges, or erroneous output at some point during peptide construction. Missed reagent charges are because they are done using level sensors. In contrast, the system can be designed to measure all reagent charges by mass from a weighing scale. A single experiment typically needs to run for more than a month, for example, if the peptide has more than 30 amino acids. An incorrect amino acid loading near the end of a month-long experiment means the experiment must be restarted, wasting a month (or the additional time required for synthesis) along with the wasted materials. This is less likely to happen with DeltaV automation, as it is designed to be more reliable and robust, being the industry standard for GMP production.

[0048] In a further embodiment, due to its online analysis, the system can be designed to provide significantly more process information and understanding than other commercially available synthesizers. Online LCMS (liquid chromatography / mass spectrometry) of peptides on resin simultaneously quantifies the conversion and kinetics of all deprotection and coupling reactions in all parallel reactors. Timing is integrated with the chemical process because the same DeltaV system running the process also performs online analysis. Therefore, staff may not need to collect and analyze samples in the laboratory to make forward processing decisions.

[0049] To obtain an online LCMS system, you can perform the following steps:

[0050] 1. Take 1.0 mL of slurry from the resin reactor.

[0051] 2. Immediately lyse the sample with TFA in a small reactor (the time from sample removal to the start of TFA lysis is approximately 2 minutes).

[0052] 3. Dilute the lysed peptide solution in LC (liquid chromatography) diluent and mix.

[0053] 4. Pass the laboratory delivery solution through the LC injection loop (no air bubbles).

[0054] 5. Switch the loop to inject the sample onto the LC.

[0055] 6. Wash the waste resin beads from the pyrolysis reactor into the waste.

[0056] 7. Clean the sample valve and tubing with solvent.

[0057] However, it should be noted that for this in-line LCMS to be implemented, the flow is vertically upward through the injection valves to ensure they are completely filled without bubbles. Two three-way valves are used to turn the slurry sample out of the sample loop and blow it into the pyrolysis and deprotection zone. A single solenoid gas delivery line is connected to the actuators of these two valves so that they switch exactly simultaneously, which may be important for obtaining exactly 1 mL of slurry per sample. The slurry then travels through the sample zone and continues upward over the sample valve. This is important so that even if the reactor viscosity and slurry density change from one step to the next and the flow distance after passing the valve changes, the sample valve is filled with a representative slurry density. The slurry sample tube from the reactor continues upward to the sample valve. This is important so that the tube is clean when later pumped back to the reactor in reverse. It minimizes entrainment and prevents solid blockage. After each sample is completed, the sample valves are flushed with solvent using dilution carts. Otherwise, they would eventually become clogged with solids. The solvent enters between the sample valve and the peristaltic pump in a backward direction away from the valve and then propels forward through the valve. Solvent slugs flush the pyrolysis zone one step at a time to remove resin solids. After passing the valve, the slurry continues to flow uphill. This uphill flow is long enough to provide sufficient margin to ensure a representative sample enters the sample loop, but the slurry does not cross the apex and begin to flow back downhill. If it did, it would reach the peristaltic pump, which can cause entrainment problems and abrade the resin, leading to filtration issues in the reactor. The peristaltic pump is located after the downhill portion of the sample loop, not upstream. When it is upstream of the sample loop, it causes entrainment problems and abrades the resin, leading to filtration issues in the reactor. A valve (which can be customized) can be used, with additional ports welded to the valve body, to allow the diluent solvent to enter directly at the top of the sphere and push upwards. This thoroughly mixes the diluent solvent with the pyrolysis solution and also disturbs the resin bed deposited at the top of the sphere.

[0058] Let's refer to it together now. Figure 1 and 2The addition of the next amino acid in the peptide sequence is now described. This next amino acid in the sequence may be designated "Z," meaning it can be any desired amino acid (as mentioned above, amino acid "X" can also be any desired amino acid). The steps and procedures outlined above are used to couple amino acid "X" to the resin. (Thus, amino acid "X" becomes the first amino acid in the peptide sequence). Once this amino acid "X" has been coupled and attached to the resin, the resin in the first, second, and / or third reactors 106, 108, 112 can be washed with a solvent. This solvent is typically the same solvent outlined above. This washing can be performed in a sequential (e.g., tandem) manner outlined herein. Thus, for example, a first additional amount of solvent may be added to the first reactor 106, stirred in the first reactor 106, then transferred to the second reactor 108 (and used to wash the resin in the second reactor 108), and then transferred to the third reactor 112. Similarly, once the first additional amount of solvent has been removed from the first reactor 106, a second additional amount of solvent may be added to the first reactor 106 (and recycled through the other reactors). (A third additional amount of solvent may also be recycled through the system in the same way.) In other words, the washing steps can be performed in the order and manner outlined above.

[0059] Once reactors 106, 108, and 112 have been washed with solvent, a first additional amount of deprotecting agent is added to the first reactor 106, and then (after a period of time) removed from the first reactor 106 and added to the second reactor 108. This first additional amount of deprotecting agent is then (after a period of time) also removed from the second reactor 108. A second additional amount of deprotecting agent is added to the first reactor 106, reacted for a period of time, then removed from the first reactor 106 and transferred to the second reactor 108, where it reacts, and then removed from the second reactor 108 (and then added to the third reactor 112 in the manner outlined herein). This first and second additional amounts of deprotecting agent (and the third additional amount of deprotecting agent), individually or collectively, function to deprotect the protected N-group of amino acid “X” in the first and second reactors.

[0060] Following this reaction with the deprotecting agent, the amino acid "X" (attached to the resin) is ready to be coupled to the next amino acid "Z" in the desired sequence. Therefore, the following steps are performed in the manner outlined herein:

[0061] The first amount of amino acid "Z" is added to the first reactor 106;

[0062] The first amount of amino acid "Z" is transferred to the second reactor 108;

[0063] A second amount of amino acid "Z" is added to the first reactor 106, wherein the first and second amounts of amino acid "Z", individually or jointly, couple amino acid "Z" to the deprotected N group of amino acid "X" in the first reactor 106.

[0064] The first amount of amino acid "Z" is removed from the second reactor 108; and

[0065] The second amount of amino acid "Z" is transferred from the first reactor 106 to the second reactor 108; and

[0066] The second amount of amino acid "Z" is removed from the second reactor 108.

[0067] The first and second amounts of amino acid "Z", individually or jointly, couple amino acid "Z" to the deprotected N group of amino acid "X" in the second reactor 108.

[0068] Those skilled in the art will recognize that amino acid "Z" can also be reacted with amino acid "X" in the third reactor 112 in a similar manner using these series of reactors and reagents (with each amino acid added sequentially). Thus, the amino acid sequence is constructed in this way. The process is then iteratively repeated, adding the next amino acid, and the next, as is known in SPPS synthesis.

[0069] Now for reference Figure 3 This image shows a schematic view of another embodiment of a system 300 for coupling an amino acid “X” to a protected N-group attached to a peptide synthesis resin. Specifically, system 300 includes a plurality of tanks 301 designed to contain a quantity of amino acids. Specifically, each specific amino acid to be added to the peptide chain may have its own individual tank 301. Furthermore, each tank 301 may have its own pump 303, designed to pump the amino acid (soluble in solution) from tank 301 to activation reactor 307. Specifically, pump 303 pumps the amino acid solution out of tank 301 via line 309, into line 311, and into reactor 307. Those skilled in the art will appreciate how such piping / lined connections and pump 303 are made to achieve this transfer of the amino acid solution from the individual tanks 301 to reactor 307. Figure 3 Only three different amino acid tanks (301) are shown. More can be used as needed.

[0070] Optionally, one or more flow sensors 313 may be connected to line 309 and / or pump 303 to sense the flow rate of amino acids passing through the line (and entering reactor 307), thereby allowing adjustment of the quantity, flow rate, flow timing, etc., as needed. Additionally, valve 317 may be used to transfer the amino acid flow back to feed container 301 to activate the pump. Those skilled in the art will appreciate the use of valves and / or inerting vent system 327, which, if necessary, may also include vent 329 and overflow container for safety reasons.

[0071] Similarly, system 300 includes multiple storage tanks 353 designed to hold quantities of other reagents, such as DIC, oxyma, and other washing solvents. Furthermore, each tank 353 may have its own pump 355, designed to pump liquid from the storage tank 353 to the activation reactor 307 or any filtration reactors 306, 308, 312. Those skilled in the art will appreciate how such piping / lined connections and pumps 355 are made to achieve this transfer. Figure 3 Only three different feed tanks (353) are displayed. More can be used as needed.

[0072] Optionally, one or more flow sensors 359 may be connected to the line from pump 355 to sense the flow rate through the line, thereby allowing adjustment of the quantity, flow rate, flow timing, etc., as needed. Additionally, valve 357 may be used to transfer the flow back to feed container 353 to start the pump. Those skilled in the art will appreciate how valves and / or inerting system 363 may be used; if necessary, inerting system 363 may also include an exhaust bubbler 361 and an overflow container for safety reasons.

[0073] As described above, system 300 includes an activation reactor 307. The activation reactor 307 is typically located upstream of the first reactor 306, the second reactor 308, and the third reactor 312. The activation reactor 307 may optionally include a stirrer 333, which is designed to mix the contents (solution) within the activation reactor 307. A temperature probe 335 may also be added to the activation reactor 307. A circulator 341 associated with a jacket 345 may also be included (optionally).

[0074] The activation reactor 307 can be designed to "activate" the amino acid solution by premixing it with DIC and oxyma. Those skilled in the art will appreciate how DIC and oxyma (and / or solvents and some other activators and bases) can be added to reactor 307.

[0075] System 300 also includes a deprotection solution container 371, which contains the deprotection reagent. (In Figure 3 In the embodiment shown, the deprotecting agent is piperidine (although other materials may also be used). One or more valves 369 and pump 365 may be designed to pump the deprotecting agent (via line 367) to the first reactor 306 (via inlet line 379). A pressure relief valve 381 may optionally be added to line 367. Additionally, a pressure sensor 385 may optionally be used within container 371. An additional valve 383 may optionally be used as part of the venting system for container 371, if desired.

[0076] A storage container 371a for the solvent (in this case, DMF or some other solvent) may also be used as part of system 300. Storage container 371a may (optionally) include a pressure sensor 373 to measure the pressure of the solvent. One or more valves 375 and a pump 377 may be used to deliver the solvent from container 371a. The solvent is delivered via line 381 to activation reactor 307, first reactor 306, second reactor 308, or third reactor 312. Flow sensor 383 may (optionally) be used to measure the flow rate through line 381. An additional vent valve 386 may be optionally used as needed.

[0077] As described in the above embodiments, the first, second, and third reactors 306, 308, and 312 contain a certain amount of resin (such as Sieber resin) and are arranged in series downstream of the activation reactor 307. For safety purposes, these reactors may include an inert headspace metered nitrogen supply 403 and one or more exhaust overflow containers 401. As those skilled in the art will appreciate, other exhaust ports and / or safety measures can be designed.

[0078] As described above, the solvent solution is connected via one or more lines 381 to each of the first, second, and third reactors 306, 308, and 312, and to the activation reactor 307. One or more valves 405 control the flow of solvent into each of these containers. In other embodiments, the solvent inlet point for each of these reactors may be a “spray ball” or other spray feature capable of efficiently introducing solvent into the container and “washing” the sides of the container (and any solids that may be present thereon), thereby ensuring that all resins are properly mixed and exposed to the reagents and washing solvent. (In fact, it is generally recommended to spray reactors 306, 308, and 312 with solvent in this manner when “washing” the resin in reactors 306, 308, and 312 between the addition of each specific amino acid).

[0079] As described above, the solution leaving the activation reactor 307 exits via an outlet bottom port and connected piping. Optionally, a flow sensor 409, one or more flow control valves 411, and a pump 413 may be present in the flow of this outlet line. Furthermore, the outlet line may include one or more valves 415, which allow automation to selectively control the flow of solution through the line so that the solution may be directed to waste 419 or to the first, second, and / or third reactors 306, 308, 312. (Typically, system 300 operates with the second and third reactors 308, 312 in series with the first reactor 306, so that the flow first enters the first reactor 306, but valves 415 (which may be three-way valves (or two-way valves or four-way valves, etc.) allow the user to control the flow and modify it as needed.)

[0080] The first, second, and third reactors 306, 308, and 312 used in this system 300 are similar to and / or identical to those described above. These reactors 306, 308, and 312 may optionally each include a stirrer 421 and a temperature sensor 423. The first, second, and third reactors 306, 308, and 312 include a resin that serves as a substrate for constructing peptides. This resin includes protected N-groups, which can be deprotected using a deprotecting agent from vessel 371, and then reacted with an activated amino acid solution (activated in reactor 307), thereby coupling this amino acid to the sequence / resin (using SPPS technology). Different amino acids from vessel 301 can be added as needed by sequentially performing these steps.

[0081] As described above, the first reactor 306, the second reactor 308, and the third reactor 312 may be arranged in series. Accordingly, a first amount of activated amino acids is reacted in the first reactor 306, and then that amount is sequentially fed to the second and third reactors 308, 312 in the manner described above (and a new amount of activated solution is added to the first reactor 306). To facilitate this flow, each reactor 306, 308, 312 may include a filter 427 to ensure that solid peptide resin beads and / or solid forming peptides are retained in the reactors 306, 308, 312. Optionally and / or as needed, flow sensors 433, valves 437, and pumps 435 may be used to guide this “tandem” flow between the first, second, and third reactors 306, 308, 312. Similarly, sampling devices 441 may be positioned anywhere in the system (or multiple sampling devices 441 as needed) to sample the material flowing through the system 300, thereby ensuring its proper operation or inspecting the waste stream to determine when washing is adequate.

[0082] It should be pointed out that, in Figure 3In one implementation, valve 437 may be designed to allow the outputs of the first, second, and third reactors 306, 308, and 312 to flow sequentially in series and / or to waste 419. This allows automation to control the desired flow. Furthermore, in Figure 3 In the diagram, the output line from the third reactor 312 is shown leading to waste 419. As described above, the implementation can be designed such that when reactors 306, 308, and 312 and / or activation reactor 307 are washed with solvent, the solvent is reused (and returned to the reactor), thereby making the system more environmentally friendly. Similarly, the activated amino acid solution can flow through the first, second, and third reactors 306, 308, and 312, and then be recycled again through the same reactor (and / or activation reactor 307) (multiple times, as needed) – as a means of re-implementing the reaction (but preferably, if desired, running the reaction in parallel). For example, when “washing” the resin between the addition of each specific amino acid, this washing is typically carried out in 10 different washing steps. If so, the first 2 to 8 washes can be performed with a pre-used solvent, as this solvent, although potentially impurities, is “clean” enough for the first few batches of washing, and then the final few washes (2 to 8 washes) are performed with pure solvent. The washing solvent from the last 2 to 8 washes is collected in a recycling container for the first 2 to 8 washes of the next cycle. This reduces the amount of solvent used throughout the washing process.

[0083] Now for reference Figure 4 The display system 500 is compatible with... Figure 1-3 The implementation scheme incorporates an online LCMS system. Specifically, system 500 is designed to measure peptide samples bound to the solution and the solid-phase resin in the aforementioned reactor, thereby providing operators with real-time information about what is happening in the system.

[0084] System 500 includes a lysis solution vessel 503. In many embodiments, this lysis solution is TFA, which is designed to cleave the peptides formed on the resin (and thus separate the formed peptides from the resin). TFA can also be used to quench coupling reactions. Valves 518p, 518q, and 518s are as follows: Figure 4 The diagram shows the control of TFA solution (or pyrolysis solution) flow into the system. A pyrolysis overflow area 507 may also be present, comprising a pipeline 508, an overflow container 509, and a measuring area 511 (as well as a valve 518r connected to a nitrogen supply 522). This system 507 is designed to measure the required amount of TFA and return any excess to the storage container 503. Those skilled in the art will appreciate how these safety features and / or flow features are implemented to control the TFA solution flow. This may involve the use of one or more valves 518.

[0085] System 500 also includes a mixing tank 510 (which may be a 500 mL container). The mixing tank 510 receives a slurry sample from the reactor solvent (which may be DMF) via a solvent line 512 controlled by valve 518a. These lines 512 (along with valves 518b and 518a) are capable of recovering slurry from the reactor (whether it is an activation reactor or the first, second, or third reactor described above). Lines 512 may have an 1 / 8-inch inner diameter and a 1 / 4-inch outer diameter. Lines 512 and valves 518a and 518b also allow the addition of gas (from nitrogen supply 522) to the system and to the pump-around loop. Valve 518a also allows material to flow to the reactor return 524 and out of the reactor 525. Valve 518b allows DMF (solvent) to be obtained from manifold 526 as needed. The purpose of line 512 is to extract a sample of the reaction slurry (e.g., 1 mL) so that it can be added to tank 510. A valve can be used to ensure that material flows out of reactor 525 (and, if necessary, upward) so that the slurry does not reach the pump, thus preventing abrasion of the resin beads. Accordingly, by switching the valve and using nitrogen, the slurry can be extracted and the remainder pumped back to reactor 525 without abrasion of the resin in the pump. Those skilled in the art will appreciate how this can be done.

[0086] In addition to the slurry sample, another feed into the mixing tank 510 comes from the diluent supply 520, which adds solvent or other materials as needed to dilute 1 ml of sample. This diluent supply 520 is connected to the mixing tank via line 528. A diluent overflow system 527 may also be used. This system 527 may also include a diluent overflow area 529, which includes line 528, an overflow container 529, and a measuring area 521 (as well as valves 518e and 518l connected to nitrogen). Valve 518h controls the flow into the overflow container 529. This diluent overflow system 527 is designed to measure the required amount of diluent and return any excess to the storage container 520. Those skilled in the art will appreciate how these safety features are implemented and how the diluent solution flow is controlled.

[0087] Once in mixing vessel 510, the lysed and diluted sample flows via line 540 to valve 518m. Line 540 delivers the material (typically a peptide lysed from resin or a grown amino acid sequence) to HPLC 544 for analysis. A storage vessel 541 (which may be a 300 mL container) can be used upstream of the HPLC to separate bubbles from the liquid by gravity and to store the sample as needed.

[0088] Mixing tank 510 can be connected to line 550 (and valve 518j) as needed to vent mixing tank 510. Sensor 551, such as a pressure or temperature sensor (or other sensor), can measure the conditions in this line (or typically in this system—if located elsewhere in system 500). Pressure sensor 551 serves as an indication in the automation sequence when to proceed to the next step.

[0089] Typically, after mixing in mixing tank 510 for 30 minutes to allow time for peptide deprotection (and dilution), the material can be extracted from mixing tank 510 via line 555 and allowed to deposit at or near valve 518k. Once valve 518k is opened, liquid and / or waste resin beads can flow into waste 546 via line 555. A caustic bubbler 557 can be used as a safety mechanism to clean any TFA that may be present in line 555. If necessary, nitrogen or other gases can be added to waste 546 via line 561 and valve 562 to facilitate this washing. Other safety features can also be added, such as a knockout pot 569. The knockout pot 569 is designed to prevent bubbler liquid from being drawn back into the system. Figure 4 As shown, this gas-liquid separator 569 can also be connected via pipeline 580 to a nitrogen supply 581 that supplies nitrogen or other gases to inert 569, 546 and 557.

[0090] Those skilled in the art will recognize that the following should be used Figure 4 The system 500 outlined herein samples the reactor at the following frequency. In some embodiments, sampling can be performed at regular intervals, such as every 45 minutes, during the coupling reaction. In other cases, sampling may be performed at other intervals, such as every 60 minutes or during the washing step. Of course, those skilled in the art can design other sampling frequencies.

[0091] In other embodiments, sampling from the activation reactor can be performed automatically before the contents of the activation reactor are added to the first reactor. This is done to verify that the amino acids added to the activation reactor (and subsequently to the first, second, and third reactors) are the desired amino acids—for example, the next amino acid in the sequence. This is because, for example, if 25 amino acids have already reacted together and the “wrong” amino acid is added, the entire peptide sequence would be incorrect, and peptide synthesis would have to start from scratch. Therefore, to prevent such errors, amino acids are automatically sampled before they are added to the coupling reaction, thereby minimizing the chance of adding the “wrong” amino acid.

[0092] Now for reference Figure 5 ,show Figure 1 Perspective views of reactors 106, 108, and 112 shown. (See diagram.) Figure 5 As can be seen, resin 120 is porous, such that some activated ester solutions (as outlined herein) can be absorbed into the pores and continue to react (as outlined herein).

[0093] Another aspect of this embodiment is that the online LCMS system can be used with the first reactor. This can be the online LCMS system described herein, used with the first reactor of a tandem reactor system of the type described herein. Other embodiments can be designed in which the online LCMS system is used with a single reactor, which in some embodiments may be a batch reactor. The advantage of this LCMS system is that it can automatically extract precise samples (e.g., 1 mL) at various times during the reaction process (e.g., at the start, middle, and / or end of the reaction). The user can program when to automatically extract samples. The extracted samples can be used to test how the reaction proceeds and whether the reaction is complete. Such sampling can be performed during activation, reaction steps, and / or washing steps. By using this LCMS, Kaiser assays (which are typically used to monitor the progress of peptide coupling) may be unnecessary (thus saving cost and time). Sampling also provides real-time monitoring of the reaction, which is particularly valuable at production scale. Although samplers such as Mettler Toledo smaplers exist, such devices are not used in SPPS reactors and do not include apparatus and methods for pyrolysis and deprotection, dilution, mixing, separation from waste resin beads, delivery to the LCMS, and storage in the LCMS switching loop. Figure 4 The detailed procedure for the automated sequence of the online LCMS system shown is as follows.

[0094] Peristaltic sampling sequence of Cart 491 peptide synthesis

[0095] Updated: December 15, 2020

[0096] Begin by venting down. Valve opens:

[0097] Open 618A, 518k [618A, 518k]

[0098] Waiting time for user input "Vent PSI Read Dly"

[0099] Wait until PT < “Vent Low”

[0100] Shut down 618A, 518k [ ]

[0101] sample

[0102] Open valve 518j [518j]

[0103] Command the sample peristaltic pump to start "forward".

[0104] Waiting for user to input time "Pump1 Time1"

[0105] Command to stop sample peristaltic pump

[0106] Connect valve 518a (three-way valve connected to sample) [518a, 518j]

[0107] Waiting for "AB open time"

[0108] Shut-off valve A (three-way valve returning to reactor / racetrack) [518j]

[0109] Command the sample peristaltic pump to change direction to "backwards".

[0110] Waiting for user input of "Pump1 CCW time"

[0111] Command to stop sample peristaltic pump

[0112] Add pyrolysis solution

[0113] Open 518r, 518s [518j, 518r, 518s]

[0114] Wait 5 seconds to pressurize the lysis solution vial.

[0115] Disable 518r, 518s [518j]

[0116] Open 518q [518j, 518q]

[0117] Wait 5 seconds to purge the pyrolysis solution measurement area.

[0118] Close 518q [518j]

[0119] Open 518p [518j, 518p]

[0120] Waiting time for user input "P Open Time"

[0121] Close 518p [518j]

[0122] Open 518r, 518s [518j, 518r, 518s]

[0123] Waiting time for user input "S Open time"

[0124] Turn off 518s [518j, 518r]

[0125] Open 518q [518j, 518q, 518r]

[0126] Waiting for the user to input the time "Q Open time"

[0127] Close 518r, 518q, [518j]

[0128] Deprotection

[0129] Waiting for "Deprotect Time"

[0130] Meanwhile waiting

[0131] Each "Mix Delay Time" (user input parameter)

[0132] Open valves L and 618A [518j, L, 618A]

[0133] Waiting for "Mixing Time" (user input parameter)

[0134] Shutdown valves L, 618A [518j]

[0135] shut-off valve 518j [ ]

[0136] Measuring diluent

[0137] Open G [G]

[0138] Waiting for the user to input the time "G Open Time"

[0139] Turn off G [ ]

[0140] Open 518e and 518h [518e, 518h]

[0141] Waiting time for user input "DEH Open Time"

[0142] Close 518h [518e]

[0143] Open 518r and 518q to pressurize the mixing tank [518e, 518q, 518r].

[0144] Wait until PT > Mix Pot Close

[0145] Disable 518r, 518q, [518e]

[0146] Open M1 and M2 [518e, M1, M2]

[0147] Wait until PT < LVS Empty / M2 Cls

[0148] Close M1 M2 [518e]

[0149] Mixed pyrolysis sample and diluent

[0150] Open valve 618a [518e, 618a]

[0151] Wait until PT > User enters setpoint "Mix Pot Close"

[0152] Shut-off valve 618a [518e]

[0153] Settling

[0154] Waiting for the user to input "settling time"

[0155] Transfer to HPLC

[0156] Open valve M1 [518e, M1]

[0157] Wait until PT < “M1 Close PSI”

[0158] Shut-off valve M1 [518e]

[0159] Empty the remaining slurry from the mixing tank.

[0160] Open 618a [518e, 618a]

[0161] Wait until PT > User enters setpoint "Mix Pot Close"

[0162] Close 618a [518e]

[0163] Open 518k [518e, 518k]

[0164] Wait until PT reads < “Vent Low”

[0165] Disable 518k [518e]

[0166] If Agilent:

[0167] The sample was stored on an HPLC.

[0168] Open 618a [518e, 618a]

[0169] Wait until PT > User inputs setpoint "mix pressure" (same setpoint as above).

[0170] Open M1 [518e, 618A, M1]

[0171] Waiting for "M3 Open Delay"

[0172] Open M3 [518e, 618A, M1, M3]

[0173] Waiting for "M3 Open Time"

[0174] Turn off M3, 618a, 518e, and M1. [ ]

[0175] Send a signal to the HPLC to extract the sample.

[0176] exhaust

[0177] Open 618a, 518q, 518k [618a, 518k, 518q]

[0178] Waiting for user input of "Vent PSI Read Dly"

[0179] Wait until PT reads < “Vent Low”

[0180] Close 518q, 618a, and 518k [ ]

[0181] Wait until the next order sample

[0182] Pump washing

[0183] This is performed after the last coupled or deprotected sample.

[0184] Connect valve 518a (three-way valve connected to the mixing tank) [518a]

[0185] Open valve 518b (DMF) [518a, 518b]

[0186] The command to run the sample peristaltic pump "forward" until the user-input time is reached.

[0187] Command to stop sample peristaltic pump

[0188] Shut-off valve 518b [518a]

[0189] Shut-off valve 518a (three-way valve return to reactor / racetrack) [ ]

[0190] Valve washing:

[0191] Command the sample peristaltic pump to run "reverse" for 1 second.

[0192] Command to stop sample peristaltic pump

[0193] Connect valve 518a (three-way valve connected to the mixing tank) [518a]

[0194] Open valve 518k [518a, 518k]

[0195] Wait 5 seconds

[0196] Close valve 518k [518a]

[0197] Shut-off valve 518a (three-way valve return to reactor / racetrack) [ ]

[0198] Repeat valve washing 3 times

[0199] The command to run the sample peristaltic pump in "reverse" mode until the user-input time is reached.

[0200] Command the sample peristaltic pump to stop.

[0201] Example 1

[0202] Construction of tirzepatide via solid-phase peptide synthesis (SPPS)

[0203] tirzepatide ( Figure 6 The tirzepatide backbone, containing 39 amino acids, was synthesized via linear solid-phase peptide synthesis (SPPS). The synthesis of tirzepatide involved first constructing a tandem reactor as described below. Figure 7 The 39-mer main chain intermediate shown is used. Note that during the synthesis of tirzepatide, the 39-mer main chain intermediate ( Figure 7 It is attached to a solid support (such as the Sieber resin described herein) via the -NH2 group attached to serine-39.

[0204] Table 1 lists the sequence of each of the 39 amino acids used to construct the tirzepatide backbone. For example, serine is the first amino acid used in the backbone synthesis; it is amino acid position 39 in tirzepatide. In linear SSPS, the nitrogen of each amino acid used from Ser39 to Aib2 is protected by a 9-fluorenemethyloxycarbonyl group (Fmoc) on the α-nitrogen, except for Tyr1, which is protected by a tert-butyloxycarbonyl group (Boc) on the α-nitrogen. For the side-chain protecting groups as shown in Table 1, oxygen is protected with a tert-butyl group (tBu), and nitrogen is protected with triphenylmethyl (Trt), 1-(4,4-dimethyl-2,6-dioxocyclohexyl-1-idel)-3-methylbutyl (ivDde), or Boc.

[0205] surface The sequence of 39 amino acids used to construct Tirzepatide via SPPS

[0206] amino acid (AA) addition order AA position on peptide AA Name AA for coupling steps 1 39 Serine Fmoc-Ser(Bu)-OH 2 38 proline Fmoc-Pro-OH 3 37 proline Fmoc-Pro-OH 4 36 proline Fmoc-Pro-OH 5 35 alanine Fmoc-Ala-OH 6 34 glycine Fmoc-Gly-OH 7 33 Serine Fmoc-Ser(Bu)-OH 8 32 Serine Fmoc-Ser(Bu)-OH 9 31 proline Fmoc-Pro-OH 10 30 glycine Fmoc-Gly-OH 11 29 glycine Fmoc-Gly-OH 12 28 alanine Fmoc-Ala-OH 13 27 Isoleucine Fmoc-Ile-OH 14 26 Leucine Fmoc-Leu-OH 15 25 Tryptophan Fmoc-Trp(Boc)-OH 16 24 glutamine Fmoc-Gln(Trt)-OH 17 23 Valine Fmoc-Val-OH 18 22 Phenylanine Fmoc-Phe-OH 19 21 alanine Fmoc-Ala-OH 20 20 Lysine-ivDde Fmoc-Lys(ivDde)-OH 21 19 glutamine Fmoc-Gln(Trt)-OH 22 18 alanine Fmoc-Ala-OH 23 17 Isoleucine Fmoc-Ile-OH 24 16 Lysine Fmoc-Lys(Boc)-OH 25 15 Aspartic acid Fmoc-Asp(Bu)-OH 26 14 Leucine Fmoc-Leu-OH 27 13 2-Aminoisobutyric acid Fmoc-Aib-OH 28 12 Isoleucine Fmoc-Ile-OH 29 11 Serine Fmoc-Ser(Bu)-OH 30 10 Tyrosine Fmoc-Tyr(Bu)-OH 31 9 Aspartic acid Fmoc-Asp(tBu)-OH 32 8 Serine Fmoc-Ser(Bu)-OH 33 7 threonine Fmoc-Thr(Bu)-OH 34 6 Phenylanine Fmoc-Phe-OH 35 5 threonine Fmoc-Thr(Bu)-OH 36 4 glycine Fmoc-Gly-OH 37 3 glutamic acid Fmoc-Glu(Bu)-OH 38 2 2-Aminoisobutyric acid Fmoc-Aib-OH 39 1 Boc-tyrosine Boc-Tyr(Bu)-OH

[0207] Series reactor method

[0208] A 20 vol% solution of piperidine in DMF was prepared as follows: Piperidine (800 mL) was diluted to a volume of 4.0 L by adding DMF to obtain a 20 vol% solution.

[0209] The following is a method for preparing a 0.68 M solution of oxyma in DMF: Ethyl cyanoacetate (oxyma, 386.85 g, 2.722 mol) was dissolved in DMF to a volume of 4.0 L to obtain a 0.68 M solution, and then nitrogen gas was bubbled through the solution at 2SCFH.

[0210] A 0.60 M solution of DIC in DMF was prepared as follows: N,N'-diisopropylcarbodiimide (340.8 g, 2.700 mol) was dissolved in DMF to a volume of 4.5 L to obtain a 0.60 M solution, and then nitrogen gas was bubbled through the solution at 2 SCFH.

[0211] The following is a sample preparation of a 0.375 M solution of serine in DMF: FMOC-Ser( t Bu)-OH (431.3 g, 1.318 mol) was dissolved in DMF until a volume of 3.0 L was reached with DMF, shaken to dissolve, and then nitrogen gas was bubbled through the solution at 2 SCFH. Similarly, 0.375 M solutions of each amino acid shown in Table 1 were prepared.

[0212] The reaction system was prepared as follows: Three reactors of equal size were equipped with filters to retain solid resin when the solution was pumped out. Reactors were labeled “RB1”, “RB2”, and “RB3”, and Sieber resin (500 g, 0.70 mmol / g, 350 mmol) was added and divided equally between the reactors. 1500 mL of DMF was added to each reactor and the mixture was stirred at room temperature for 24 hours to allow the resin to swell.

[0213] General Program A - F-moc Deprotection and DMF Washing ProcessAdd piperidine solution (20 vol% in DMF, 1365 mL) to RB1 and stir at room temperature for 30 min. Pump the piperidine solution from RB1 to RB2, then load another portion of piperidine solution (20 vol% in DMF, 1386 mL) into RB1 and stir reactors RB1 and RB2 for 30 min. At the end of the stirring time, pump the piperidine solution from RB2 to RB3, pump the piperidine solution from RB1 to RB2, then load another portion of piperidine solution (20 vol% in DMF, 1407 mL) into RB1. Stir all three reactors at room temperature for 30 min. Pump the piperidine solution from RB3 to waste, then pump the piperidine solution from RB2 to RB3, then pump the piperidine solution from RB1 to RB2. Stir RB2 and RB3 at room temperature for 30 min. Pump the piperidine solution from RB3 to waste, then pump the piperidine solution from RB2 to RB3. Stir RB3 at room temperature for 30 min, then pump the piperidine solution from RB3 to waste. In this way, the piperidine solution was pumped through three reactors in series three times, with each reactor stirred for 30 minutes.

[0214] After all three fractions of piperidine solution had been stirred through all three reactors and sent to waste collection, DMF (1218 mL) was added to RB1 and stirred at room temperature for 5 min. This solvent was then pumped from RB1 to RB2, and DMF (1208 mL) was added to RB1. Both containers were stirred at room temperature for 5 min. This solvent was then pumped from RB2 to RB3 and from RB1 to RB2. DMF (1758 mL) was added to RB1, and all three containers were stirred at room temperature for 5 min. This same procedure was repeated until fractions of DMF solvent had been pumped through the three reactors in series (with DMF solvent pumped from RB3 to waste each time), with a total of 8 washes per reactor, using a total of 12.66 L (11.95 kg) of DMF. The average DMF wash volume was 1580 mL. The target wash volume was 1400 mL per wash, therefore, for subsequent amino acids in peptide synthesis, the pump and feed tank pressures were adjusted to bring subsequent loads close to 1400 mL.

[0215] General Procedure B – Amino Acid Activation and Coupling ProcessA serine solution (0.375 M in DMF, 774 g), an oxyma solution (0.68 M in DMF, 422 g), and then a DIC solution (0.60 M in DMF, 506 g) were added to a jacketed reactor labeled “RA”. The solution was stirred at 15°C for 30 min to obtain an activated serine solution, which was then added to RB1. This process was repeated to prepare an activated serine solution in reactor RA and add it to RB2, and then the process was repeated again to add the activated serine solution to RB3. Each of the three reactors had a total slurry volume of approximately 2200–2300 mL during the coupling reaction, which is equal to the volume of the activated ester solution plus the swollen resin. Reactors RB1, RB2, and RB3 were stirred at room temperature for 8 hours, and then the solvent was drained from all three reactors.

[0216] Reactors RA, RB1, RB2, and RB3 were washed in series with DMF solvent in a manner similar to General Procedure A, with each reactor washed a total of 7 times. The first 3 washes were pumped into the spray ball in RA before being pumped into RB1. Therefore, the first 3 washes were performed in series through RA, RB1, RB2, and RB3. However, each time the wash solution was pumped out of RA, a small portion of the solvent wash solution was pumped from RA to RB3 (~30 mL), then a small portion of the solvent wash solution was pumped from RA to RB2 (~30 mL), and then the majority of the solvent was pumped to RB1 (~1340 mL). The piping between RA and RB3 and between RA and RB2 was then evacuated. This flushed and emptied the transfer piping and valves between RA and each resin reactor. Then, for the 4th through 7th washes, a small portion of DMF solvent was pumped through the spray ball in RB3 (100 mL), then a small portion of DMF solvent was pumped through the spray ball in RB2 (100 mL), and then 1400 mL of DMF solvent was pumped through the spray ball in RB1. The purpose was to flush away solid resin particles from the walls of each reactor. The total amount of solvent used for washing after coupling was 10.69 L (10.09 kg). For later amino acids in the synthesis of the 39mer peptide backbone, the amount of DMF sprayed into RB2 and RB3 via the spray ball was reduced to ~50 mL.

[0217] For each amino acid listed in Table 1, from proline 38 to Boc-tyrosine 1, Fmoc deprotection of the previously coupled amino acid was performed using Universal Procedure A, followed by coupling using Universal Procedure B. For the coupling of serine 39 and the next 38 amino acids, Universal Procedure B was used, with stoichiometry listed in Table 2. When indicated in Table 2, additional activated esters were prepared according to Universal Procedure B with the stoichiometry given in Table 2 and then recoupled. The coupling reaction was stirred until >99% conversion was achieved.

[0218] Table 2. Molar equivalents of amino acids, oxyma, and DIC relative to resin in each reactor.

[0219]

[0220]

[0221]

[0222]

[0223] After coupling, perform an average of 10 DMF washes (see Table 8). During manufacturing runs, 7 washes are recommended to save time and solvent.

[0224] Automated sample extraction for online LCMS began with proline coupling at position 37. Automated samples containing solid resin were extracted from RB3 at 15, 75, 180, 324, 503, and 684 minutes before the coupling step. Automated sample extraction from RB3 was also performed at various times during the deprotection step. For each sample, 1.0 mL of slurry sample was extracted from the reactor via an automated pump and valve. The sample was diluted with 25 mL of TFA and intermittently mixed for 30 minutes to cleave the peptide from the resin beads and remove the protecting group from the peptide. The sample was then mixed with 75 mL of 4:1 DMSO:acetonitrile. This diluted solution was stored in a 2 µL LC switching loop and injected onto the column.

[0225] Adjust the number of washes and stirring time with 20% piperidine / DMF to achieve a conversion of ≥99% as determined by LCMS. For Ser39 to Leu26, the stirring time with 20% piperidine / DMF is 30 minutes; for Trp25 and Gln24, it is 40 minutes; and for Val23 to Leu14, it is 50 minutes. For Aib13, five washes with stirring for 50 minutes each, followed by three washes with stirring for 90 minutes each. For the remaining amino acids (Ile12 to Boc-Tyr1), the first three washes with 20% piperidine / DMF are stirred for 20–30 minutes, followed by a longer stirring time during the fourth piperidine wash before entering the first reactor in series. For Pro36 and Gly34, additional deprotection and washing steps are performed as outlined in General Procedure A. It should be noted that three washes with 20% piperidine / DMF for 30 minutes each are generally sufficient to achieve a conversion of ≥99%, which will save time, solvents, and reagents in the manufacturing run of this procedure.

[0226] The number of DMF washes after the piperidine deprotection step is adjusted to ensure that the wash stream contains <600 ppm of piperidine as determined by gas chromatography (GC) before the coupling process with the next amino acid. The average number of washes used after the piperidine deprotection step is 11 (see Table 7). This is typically achieved with 10 washes, therefore 10 DMF washes after the piperidine deprotection step are recommended for manufacturing runs.

[0227] Table 3 shows the online LCMS sampling time and calculated % conversion rate throughout the synthesis of the 39-mer peptide backbone. Figure 4 Displays the process and instrumentation flow charts for automated sampling, lysis, deprotection, dilution, and storage of carts in the online LCMS.

[0228] Table 3. Online LCMS Samples and % Conversion Rate of Reactions

[0229] sample % conversion rate 75 min coupling Pro 37 94.5 180 min coupling Pro 37 99 300 min coupling Pro 37 99.8 498 min coupling Pro 37, extraction during washing process 99.6 Deprotection Pro 37 after 8 30-minute piperidine washes 99.8 75 min coupling Pro 36 89.8 180 min coupling Pro 36 99.1 480 min coupling Pro 36 99.6 After re-coupling with an additional 1 eq 200 min 99.6 After recoupling with an additional 1 eq 216 min, samples were taken during the washing process. 100 Deprotection Pro 36 after 8 30-minute piperidine washes 99.7 75 min coupling Ala 35 70.9 280 min coupling Ala 35 99.2 After re-coupling with an additional 1 eq 121 min 99.3 After recoupling with an additional 1 eq 243 min 99.2 Deprotection of Ala 35 after three 30-minute piperidine washes 99.4 After the fourth 30-minute piperidine wash followed by a 15-minute deprotection treatment, Ala 35... 99.8 After undergoing the sixth 30-minute piperidine wash followed by a 15-minute deprotection process, Ala 35... 99.9 Deprotection of Ala 35 after 6 washes with piperidine for 30 minutes each 100 15 min coupling Gly 34 36.6 75 min coupling Gly 34 65.5 180 min coupling Gly 34 98.7 360 min coupling Gly 34 99.3 After re-coupling with an additional 1 eq 15 min 100 After re-coupling with an additional 1 eq 60 min 100 After the third 30-minute piperidine wash followed by a 15-minute deprotection Gly 34 99.5 240 min coupling Ser 33 100 360 min coupling Ser 33 99.7 453 min coupling Ser 33 99.6 466 min coupling with Ser 33, sample during washing process 99.9 120 min coupling Ser 32 99.2 240 min coupling Ser 32 98.9 360 min coupling Ser 32 100 After re-coupling with an additional 0.5 eq for 15 min 100 After re-coupling with an additional 0.5 eq for 60 min 100 After re-coupling with an additional 0.5 eq for 110 min 100 After recoupling with an additional 0.5 eq for 2 h, the sample during the washing process... 100 After the third 30-minute piperidine wash followed by a 15-minute deprotection process, Ser 32 99.3 After the fifth 30-minute piperidine wash followed by a 15-minute deprotection process, Ser 32 99.4 Deprotection of Ser 32 after six 30-minute stirring cycles with piperidine 99.3 60 min coupling Pro 31 74.6 110 min coupling Pro 31 93.2 180 min coupling Pro 31 99.8 240 min coupling Pro 31 99.7 Entering the Pro 31 for the third time, 30 minutes of protection was removed and 15 minutes were left unprotected. 99.5 Entering the Pro 31 for the 5th time, 30 minutes of protection was removed and 15 minutes were left unprotected. 99.3 Deprotection of Pro 31 after 8 cycles of 30-minute stirring with piperidine 99.5 60 min coupling Gly 30 55.6 120 min coupling Gly 30 70 180 min coupling Gly 30 101.9 240 min coupling Gly 30 98.2 360 min coupling Gly 30 98.4 After recoupling with an additional 1 eq for 85 min, samples were taken during the washing process. 99.7 Entering Gly 30 for the third time, protection was removed for 15 minutes after 30 minutes. 99 Entering Gly 30 for the 5th time, protection was removed for 15 minutes after 30 minutes. 99.5 Samples were taken during the washing process of deprotected Gly 30 after six 30-minute stirring cycles with piperidine. 99.7 60 min coupling Gly 29 45.1 120 min coupling Gly 29 74.1 180 min coupling Gly 29 98.3 240 min coupling Gly 29 98.8 360 min coupling Gly 29 99.2 After recoupling with an additional 1 eq 167 min, samples were taken during the washing process. 99.4 Entering Gly 29 for the third time, protection was removed for 15 minutes after 30 minutes. 99.5 Entering Gly 29 for the 5th time, protection was removed for 15 minutes after 30 minutes. 99.4 Deprotection of Gly 29 after 8 cycles of 30-minute stirring with piperidine 99.6 60 min coupling Ala 28 52.4 120 min coupling Ala 28 78.2 180 min coupling Ala 28 98.1 240 min coupling Ala 28 98.4 360 min coupling Ala 28 98.9 After re-coupling with an additional 1 eq for 120 min 99.4 After re-coupling with an additional 1 eq for 180 min, extraction was performed during the washing process. 99.6 Entering Ala 28 for the 5th time, protection was removed for 15 minutes after 30 minutes. 99.9 Deprotection of Ala 28 after 8 cycles of 30-minute stirring with piperidine 99.9 60 min coupling Ile 27 72 120 min coupling Ile 27 91.4 180 min coupling Ile 27 97.7 240 min coupling Ile 27 98.6 360 min coupling Ile 27 99.1 Entering Ile 27 for the third time, 30 minutes later, protection was removed for 5 minutes. 99.8 Entering Ile 27 for the 5th time after 30 minutes, the protection was removed for 5 minutes. 100 Deprotection of piperidine after 8 cycles of 30-minute stirring, Ile 27 100 75 min coupling Leu 26 74.6 135 min coupling Leu 26 84.8 Entering Leu 26 for the third time, 30 minutes later, protection was removed for 5 minutes. 101.4 75 min coupling Trp 25 100 135 min coupling Trp 25 99.8 195 min coupling Trp 25 100 255 min coupling Trp 25 100 360 min coupling Trp 25 100 Trp 25 coupling for 391 min, sample during washing process. 100 Entering Trp 25 for the second time after 40 minutes, protection was removed for 5 minutes. 97.4 Entering Trp 25 for the third time, 40 minutes of protection deactivation, 5 minutes. 99.2 Entering Trp 25 for the 5th time after 40 minutes, protection was removed for 5 minutes. 98.7 Deprotected Trp 25 after six 40-minute stirring cycles with piperidine 100.1 75 min coupling Gln 24 93.6 135 min coupling Gln 24 98.6 195 min coupling Gln 24 99.9 255 min coupling Gln 24 100 360 min coupling Gln 24 100 75 min coupling Val 23 93.8 135 min coupling Val 23 99.2 195 min coupling Val 23 99.2 255 min coupling Val 23 99.5 350 min coupling Val 23 99.5 After re-coupling with an additional 1.5 eq for 60 min 99.6 After recoupling with an additional 1.5 eq for 221 min, extraction was performed during the washing process. 99.7 Entering Val 23 for the second 50 minutes, the protection was removed for 5 minutes. 95.8 Entering Val 23 for the third time, 5 minutes after the 50-minute mark, the protection was removed. 100.3 Entering Val 23 for the fourth time at 50 minutes, the protection was removed for 5 minutes. 100 Deprotection Val 23 after five 50-minute stirring cycles of piperidine 100 75 min coupling Phe 22 98.9 135 min coupling Phe 22 99.1 195 min coupling Phe 22 99.5 255 min coupling Phe 22 99.4 360 min coupling Phe 22 99.7 Phe 22 coupling after an additional 1.5 eq 120 min of re-coupling 99.8 Entering Phe 22, the second 50-minute protection was removed for 5 minutes. 94.8 Entering Phe 22 for the third time, 5 minutes after the 50-minute mark, the protection was removed. 99.5 Entering Phe 22, the fourth 50-minute protection was removed and the protection was off for 5 minutes. 100 Deprotection of Phe 22 after five 50-minute stirring cycles with piperidine 100 75 min coupling Ala 21 78.2 135 min coupling Ala 21 96.2 195 min coupling Ala 21 99 255 min coupling Ala 21 99.5 360 min coupling Ala 21 99.8 Ala 21 coupling after an additional 1.5 eq 120 min of re-coupling 99.7 Ala 21 coupling after an additional 1.5 eq 216 min of re-coupling 99.8 Deprotection of Ala 21 after five 50-minute stirring cycles with piperidine 100 75 min coupling Lys-ivDde 20 73 135 min coupling Lys-ivDde 20 82.4 195 min coupling Lys-ivDde 20 92 255 min coupling Lys-ivDde 20 95.1 360 min coupling Lys-ivDde 20 98 448 min coupling Lys-ivDde 20 98.3 565 min coupling Lys-ivDde 20 99.1 After using an additional 1.5 eq for 60 min, Lys-ivDde 20 coupling 99 Deprotected Lys-ivDde 20 after five 50-minute stirring cycles with piperidine 98.1 75 min coupling Gln 19 65.5 135 min coupling Gln 19 79.7 195 min coupling Gln 19 90.7 255 min coupling Gln 19 96.2 360 min coupling Gln 19 98.9 Gln 19 coupling after an additional 1.5 eq 60 min of re-coupling 99.4 After re-coupling with an additional 1.5 eq for 180 min, Gln 19 was extracted during the washing process. 99.6 Entering Gln 19 for the second time, 5 minutes after the 50-minute protection was removed. 93.8 Entering Gln 19 for the third time, 5 minutes after 50 minutes of protection deactivation. 99.1 Entering Gln 19 for the 4th time after 50 minutes, protection was removed for 5 minutes. 99.6 Deprotected Gln 19 after five 50-minute stirring cycles with piperidine 99.8 75 min coupling Ala 18 81.8 135 min coupling Ala 18 97.1 195 min coupling Ala 18 99.1 255 min coupling Ala 18 99.5 360 min coupling Ala 18 99.7 Ala 18 coupling after an additional 1.5 eq for 60 min 99.8 After coupling with Ala 18 for an additional 1.5 eq for 180 min, extraction was performed during the washing process. 99.9 After coupling with Ala 18 for an additional 1.5 eq for 180 min, extraction was performed during the second co-washing process. 99.9 Entering Ala 18 for the second time, 5 minutes after the protection expires (5 minutes). 87.2 Entering Ala 18 for the third time, 5 minutes after 50 minutes of protection deactivation. 98.5 Entering Ala 18 for the fourth time, 5 minutes after 50 minutes of protection deactivation. 99.6 Deprotection of Ala 18 after five 50-minute stirring cycles with piperidine 99.7 75 min coupling Ile 17 68.6 135 min coupling Ile 17 85.7 195 min coupling Ile 17 91.7 255 min coupling Ile 17 93.9 360 min coupling Ile 17 96.5 Ile 17 coupling after an additional 1.5 eq 60 min of re-coupling 98.3 After re-coupling with an additional 1.5 eq for 180 min, Ile 17 was extracted during the washing process. 99.2 Ile 17 coupling after a second recoupling with an additional 1.5 eq and 60 min. 99.6 Ile 17 coupling after a second recoupling with an additional 1.5 eq for 600 min 99.5 After a second re-coupling with an additional 1.5 eq for 689 min, Ile 17 was extracted during the washing process. 99.8 Entering Ile 17 for the first 50 minutes, then unprotecting for 5 minutes. Entering Ile 17 for the second 50 minutes, the protection was removed for 5 minutes. 90.3 Entering Ile 17 for the third time, 5 minutes after 50 minutes of protection deactivation. 99.5 Entering Ile 17 for the fourth time, 5 minutes after 50 minutes of protection deactivation. 99.7 Deprotection of Ile 17 after five 50-minute stirring cycles with piperidine 99.9 75 min coupling Lys 16 82.3 135 min coupling Lys 16 91.2 195 min coupling Lys 16 96.5 255 min coupling Lys 16 99 360 min coupling Lys 16 99.5 Lys 16 coupling after an additional 1.5 eq 60 min of re-coupling 99.7 Lys 16 coupling after an additional 1.5 eq 180 min of re-coupling 99.8 Entering Lys 16 for the second time, 5 minutes after the protection expires (5 minutes). 94.4 Entering Lys 16 for the third time, 5 minutes after 50 minutes of protection being removed. 99.7 Entering Lys 16 for the 4th time at 50 minutes, protection was removed for 5 minutes. 99.8 Deprotected Lys 16 after five cycles of 50-minute stirring with piperidine 100 75 min coupling Asp 15 89.6 135 min coupling Asp 15 98.7 195 min coupling Asp 15 99.2 255 min coupling Asp 15 99.5 360 min coupling Asp 15 99.6 After coupling with an additional 1.5 eq for 120 min, the Asp 15 coupling was performed. 99.7 After re-coupling with an additional 1.5 eq for 360 min, Asp 15 was extracted during the washing process. 99.7 Entering ASP 15 for the second time after 50 minutes, the protection was removed for 5 minutes. 99.2 Entering ASP 15 for the third time after 50 minutes of protection being removed (5 minutes). 99.8 75 min coupling Leu 14 80 135 min coupling Leu 14 93.9 195 min coupling Leu 14 99.2 255 min coupling Leu 14 99.5 360 min coupling Leu 14 99.6 Entering Leu 14 for the second 50 minutes, the protection was removed for 5 minutes. 60.1 Entering Leu 14 for the third time, 5 minutes after the 50-minute mark, the protection was removed. 95.6 Entering Leu 14 for the fourth time, 5 minutes after 50 minutes of protection being removed. 99.5 Deprotection of Leu 14 after five 50-minute stirring cycles with piperidine 99.8 75 min coupling Aib 13 66.4 135 min coupling Aib 13 79.9 195 min coupling Aib 13 88.3 255 min coupling Aib 13 89.4 360 min coupling Aib 13 94.5 474 min coupling Aib 13 96.4 Aib 13 coupling after an additional 1.5 eq 60 min of re-coupling 97.9 Aib 13 coupling after an additional 1.5 eq 180 min of re-coupling 98.1 Aib 13 coupling after an additional 1.5 eq 440 min of re-coupling 98.6 Aib 13 coupling after an additional 1.5 eq 600 min of coupling 99.3 Aib 13 coupling after an additional 1.5 eq 817 min of re-coupling 99.4 Aib 13 coupling after an additional 1.5 eq 1059 min of coupling 99.4 Entering the second 50-minute protection deprotection phase of Aib 13, 5 minutes later. 97 Entering the third 50-minute protection cycle of Aib 13, 5 minutes after protection was removed. 96.9 Entering the fourth 50-minute protection cycle of Aib 13, 5 minutes of protection was removed. 97.9 Enter the 6th stage of deprotection (90 minutes of stirring) for 5 minutes. 99.4 Enter the 7th stage of deprotection (90 minutes of stirring) for 5 minutes. 99.2 Enter the 8th stage of deprotection (90 minutes of stirring) for 5 minutes. 99.7 Deprotection of Aib 13 after 8 cycles of piperidine stirring 99.7 75 min coupling Ile 12 54.9 135 min coupling Ile 12 58.8 195 min coupling Ile 12 62.8 360 min coupling Ile 12 70.7 600 min coupling Ile 12 79.1 897 min coupling Ile 12 (entering recoupling 60 min) 86.7 1137 min coupling Ile 12 (entering recoupling 5 h) 89.9 1437 min coupling Ile 12 (entering recoupling 10 h) 94 1837 min coupling Ile 12 (entering recoupling 16.7 h) 96.7 2337 min coupling Ile 12 (entering recoupling 25 h) 98.1 2789 min coupling Ile 12 (entering recoupling 32.5 h) 99 2946 min coupling Ile 12 (entering secondary re-coupling 15 min) 99 3006 min coupling Ile 12 (entering secondary recoupling 75 min) 99.1 3171 min coupling Ile 12 (entering secondary recoupling 240 min) 99.1 3454 min coupling Ile 12 (entering secondary recoupling 523 min) 99.3 Entering Ile 12 for the third time, 20 minutes later, protection was removed for 5 minutes. 100.4 Entering Ile 12 for the 4th time after deprotection for 2 hours 99.6 The fourth deprotection of Ile 12 is nearing its end. 100 75 min coupling Ser11 97.2 135 min coupling Ser11 99.1 Entering Ser11 for the third time, protection was removed for 5 minutes after 20 minutes. 100 Entering Ser11 for the 4th time after 45 minutes of unprotection. 99.6 Final deprotected sample extracted during the washing process 99.7 75 min coupling Tyr10 93.7 135 min coupling Tyr10 98.3 195 min coupling Tyr10 99.3 255 min coupling Tyr10 99.5 360 min coupling Tyr10 100 Sample 589 min, followed by re-coupling for 60 min, Tyr10 coupling. 100 Samples were washed after 648 min of washing. Tyr10 coupling was completed. 100 Entering Tyr10 for the third time, protection was removed for 5 minutes after 20 minutes (76 minutes). 99.4 The fourth time Tyr10 was deprotected was 45 minutes (151 min). 99.5 The fourth time Tyr10 was deprotected, 2 hours (226 minutes) later. 99.7 The fourth time Tyr10 was deprotected was 4 hours (346 minutes). 99.7 The fourth time Tyr10 was deprotected, 6 hours (466 minutes) later. 99.7 The fourth time Tyr10 was deprotected for 8 hours (586 minutes). 99.7 Deprotection of Tyr10, sample during washing process (668 min) 100 15 min coupling Asp 9 90.1 75 min coupling Asp 9 98.1 135 min coupling Asp 9 99.3 195 min coupling Asp 9 99.3 255 min coupling Asp 9 99.4 360 min coupling Asp 9 99.6 562 min sample, followed by 60 min re-coupling, Asp9 coupling. 99.6 Samples were washed after 682 min of washing. Asp9 coupling ended. 99.6 851 min sample, Asp9 coupling ended, sample during washing process 99.7 Entering Asp9 for the fourth time, protection was deactivated for 2 minutes (71 min) after 5 minutes. 100 The fourth time Asp9 was deprotected occurred 1 hour (129 minutes). 99.6 Asp9 deprotection was completed, and samples were taken during the washing process (221 min). 99.6 75 min coupling Ser8 96.3 135 min coupling Ser8 97.9 195 min coupling Ser8 99.7 255 min coupling Ser8 99.5 360 min coupling Ser8 99.6 562 min sample (60 min into recoupling) Ser8 99.7 682 min sample (entering recoupling 180 min) Ser8 99.7 Entering Ser8 for the second 30-minute protection period, then unprotecting for 5 minutes (60min). 99.9 Entering Ser8 for the third time, protection was deactivated for 5 minutes after 30 minutes (105 min). 99.7 Entering Ser8 for the fourth time, protection was deactivated for 5 minutes after 30 minutes (149 min). 99.8 The fourth time Ser8 was deprotected, 2 hours (264 minutes) later. 99.8 The fourth time Ser8 protection was deactivated, 4 hours (384 min). 99.9 Ser8 deprotection complete, sample during washing process (620 min) 99.9 75 min coupling Thr7 99.7 135 min coupling Thr7 99.5 195 min coupling Thr7 99.7 Entering Thr7, the second 30-minute protection deprotection period lasted 5 minutes (65 min). 98.7 Entering Thr7 for the third time, protection was deactivated for 5 minutes (115 min) after 30 minutes. 99.7 Entering Thr7, the fourth 30-minute protection deactivation lasted 5 minutes (164 min). 100 The fourth deprotection event at Thr7 lasted 2 hours (279 minutes). 100 Thr7's final deprotection occurs during the washing process (453 min). 100 75 min coupling Phe6 96.7 135 min coupling Phe6 97.6 195 min coupling Phe6 99.1 255 min coupling Phe6 99.5 360 min coupling Phe6 99.3 485 min coupling Phe6 99.5 605 min coupling Phe6 99.2 725 min coupling Phe6 99.5 845 min coupling Phe6 99.5 903 min coupling with Phe6, sample during washing process 99.5 Entering Phe6 for the third time, protection was deactivated for 5 minutes (81 min) after 30 minutes. 98.7 Entering Phe6 for the fourth time, protection was deactivated for 25 minutes (136 min). 99.6 The fourth deprotection episode in Phe6 lasted 2 hours (230 min). 99.6 The fourth deprotection episode in Phe6 occurred 4 hours (350 min). 99.5 The final deprotection of Phe6 occurred during the washing process (441 min). 99.5 75 min coupling Thr5 99.3 135 min coupling Thr5 98.5 195 min coupling Thr5 99.3 255 min coupling Thr5 99.5 360 min coupling Thr5 99.5 486 min coupling Thr5 99.5 606 min coupling Thr5 99.6 726 min coupling Thr5 99.7 783 min coupling Thr5 during the washing process 99.7 The third 30-minute protection cycle at Thr5 lasted 5 minutes (81 min). 99.9 The fourth 30-minute protection cycle at Thr5 was followed by a 25-minute (136 min) deprotection. 99.2 The fourth deprotection of Thr5 occurred 2 hours (230 min). 99.5 The fourth deprotection of Thr5 occurred 4 hours (350 min). 99.8 Thr5's final deprotection occurs during the washing process (804 min). 100 195 min coupling with Gly4 99.2 255 min coupling Gly4 99.1 360 min coupling Gly4 99.3 486 min coupling Gly4 99.4 606 min coupling Gly4 99.5 726 min coupling Gly4 99.4 846 min coupling Gly4 99.4 966 min coupling Gly4 99.4 1238 min coupling Gly4 99.4 1358 min coupling Gly4 99.6 1478 min coupling with Gly4 99.5 1598 min coupling Gly4 99.6 1749 min coupling with Gly4 99.6 Entering Gly4 for the third time, protection was deactivated for 5 minutes after 30 minutes (81 min). 99.7 Entering Gly4 for the fourth time, protection was removed for 25 minutes (136 min). 100 The fourth time Gly4 was deprotected, 2 hours (230 min). 100 The fourth time Gly4 was deprotected, 4 hours (350 min). 100 The fourth deprotection period in Gly4 lasted 6 hours (466 min). 100 Gly4's final deprotection occurred during the washing process (588 min). 100 75 min coupling Glu3 94.6 135 min coupling with Glu3 98.6 195 min coupling Glu3 98.6 255 min coupling Glu3 99.1 360 min coupling Glu3 99 486 min coupling Glu3 99.3 606 min coupling Glu3 99 726 min coupling Glu3 99.2 846 min coupling Glu3 99.2 965 min coupling Glu3 99.4 1304 min coupling Glu3 99.4 Entering the third 30-minute protection cycle of Glu3, the protection was removed for 5 minutes (81 min). 99.8 Entering the 4th 30-minute protection cycle of Glu3, the protection was removed for 25 minutes (136 min). 99.4 The fourth deprotection of Glu3 occurred 2 hours (230 min). 100 The fourth deprotection of Glu3 occurred 4 hours (350 min). 100 75 min coupling Aib2 95 135 min coupling Aib2 99.4 195 min coupling Aib2 99.6 255 min coupling Aib2 99.7 360 min coupling with Aib2 99.7 Entering the third 30-minute protection cycle of Aib2, protection was deactivated for 5 minutes (81 min). 100 Entering the fourth 30-minute protection cycle of Aib2, protection was removed for 25 minutes (136 min). 99.6 The fourth deprotection episode of Aib2 occurred 2 hours (230 min). 100 The fourth time Aib2 protection was deactivated was 4 hours (350 min). 100 The fourth deprotection episode of Aib2 occurred 6 hours (470 min). 100 Deprotection of Aib2, sample during washing process (559 min) 100 75 min coupling with Boc-Tyr1 86.8 135 min coupling with Boc-Tyr1 94.8 195 min coupling with Boc-Tyr1 98 255 min coupling with Boc-Tyr1 99.4 360 min coupling with Boc-Tyr1 99.6 485 min coupling with Boc-Tyr1 100

[0230] Several improvements were made to the online LCMS automated sampling system for the 39mer peptide synthesis process to address solid clogging and entrainment issues. From Pro37 to Ser33, sampling was achieved using a peristaltic pump located between the reactor and the three-way sampling valve. However, this pump abraded the resin, generating fine solids, which resulted in a slow filtration rate when emptying the liquid from RB3. The resin sample pumping time was reduced, and the pump was moved after the three-way valve so that resin did not actually enter the pump. During Ala21 coupling and all subsequent couplings, the system was modified to allow flushing of both automated three-way slurry sample valves with DMF solvent after each sample. To prevent the three-way sample valves from being clogged by solids not flushed back to the reactor at the end of each sequence, DMF was flushed through the sample valves into the deprotection mixing tank and then out through valve 518k into the waste. Figure 4 ).

[0231] During the synthesis process, resin samples were taken out periodically for analysis as described above, and also for further offline studies. The amount of resin remaining in each reactor, expressed in mmol based on mass balance, is listed in Table 4. Accordingly, the amount of material loaded into the system for deprotection, post-deprotection DMF washing, amino acid coupling, and post-coupling DMF washing was adjusted based on the amount of resin remaining at each step. It should be noted that during manufacturing runs, it is unlikely that large samples will be extracted for offline analysis and the resin mmol basis will not change significantly.

[0232] Table 4. Resin mmol in each reactor after sample removal during the entire 39mer peptide synthesis process, calculated from mass balance.

[0233]

[0234]

[0235] The mass of the amino acids and coupling reagent solutions used in the amino acid coupling step is shown in Table 5.

[0236] Table 5. Mass of amino acid solution and coupling reagent used in the amino acid coupling step

[0237]

[0238]

[0239]

[0240] The piperidine concentrations obtained by GC after a series of DMF washes following piperidine deprotection are shown in Table 6.

[0241] Table 6. Piperidine concentration determined by GC in DMF-washed samples after piperidine deprotection.

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248] *BQL – Below the limit of quantitation.

[0249] In the 39-mer main chain intermediate ( Figure 7 The number and mass of piperidine solution washing and DMF washing used for deprotection throughout the synthesis process are shown in Table 7. The calculation of the mass (g) of these solutions used per millimole of resin (determined by dividing the mass by the number of millimoles of resin present in each step as listed in Table 4) is also in Table 7.

[0250] Table 7. Number of washes and reagent / solvent mass per millimolar of resin used for all deprotection processes during the synthesis of the 39-mer main chain intermediate.

[0251]

[0252]

[0253] In the 39-mer main chain intermediate ( Figure 7The mass (g) of coupling reagent used per millimole of resin throughout the synthesis process, the number of DMF washes after amino acid coupling, and the mass (g) of DMF per millimole of resin used in these washes are shown in Table 8. As in Table 7, the mass (g) of material used per millimole of resin in Table 8 is determined by dividing the mass by the number of millimoles of resin present in each step as listed in Table 4.

[0254] Table 8. Number of washes used for all amino acid couplings and reagent / solvent mass per millimolar of resin during the synthesis of the 39-mer main chain intermediate.

[0255]

[0256]

[0257] Table 9 shows the intermediates of the 39-amino acid main chain ( Figure 7 The total amount of materials loaded into the reaction vessel during the synthesis of ) is shown in Table 9. Table 9 includes the total amounts used for piperidine / DMF deprotection and subsequent DMF washing, amino acid coupling step and subsequent DMF washing, and Sieber resin.

[0258] Table 9. Total Material Mass / mmol Resin

[0259] Process steps Total mass of materials used / mmol resin (g / mmol) Piperidine / DMF solution used in the deprotection step 898.93 DMF used in washing after piperidine deprotection 2045.57 The amino acids, oxyma, and DIC solutions used in the coupling step 601.87 DMF used in washing after amino acid coupling 1871.32 Sieber resin 1.43 Total mass of materials used / mmol resin (g / mmol) 5419.12

[0260] Following Boc-Tyr1 coupling, the resin-bound peptides were removed from all three reactors, deswollen by washing with dichloromethane, and dried. The remaining steps were performed manually in batch containers, rather than in an automated tandem reactor. During the manufacturing run, resin was not removed from the reactors at this point and the subsequent steps were carried out in a tandem reactor. This reduced the amount of reagents and solvents required.

[0261] To remove the -ivDde protecting group from Lys20, hydrazine hydrate (5.34 g, 68.3 mmol) and DMF (165 g) were combined under nitrogen in a 2-liter jacketed filter reactor, and then dried resin (50.0 g, 7.24 mmol) was added. The mixture was stirred at 20°C for 6 hours, and then the resin was washed with DMF (5 × 500 mL; a wash volume of ~100 mL is recommended) with stirring for 5 minutes after each wash.

[0262] For fatty acid / connector coupling, activated esters were prepared by loading an acid of formula I (3,6,12,15-tetraoxa-9,18-diazatocetanoic acid, 22-{[20-(1,1-dimethylethoxy)-1,20-dioxoeicosyl]amino}-10,19-dioxo-,2,3-(1,1-dimethylethyl) ester, (22S); 9.5 g, 11 mmol), benzotriazol-1-yl-oxytripyrrolidone phosphonium hexafluorophosphate (PYBOP; 5.7 g, 11 mmol) and DMF (50 g) into a 250 mL round-bottom flask.

[0263]

[0264] The mixture was stirred for 20 minutes to dissolve, and then 2,4,6-trimethylpyridine (1.29 g, 10.6 mmol) was added. The activated ester mixture was stirred at 13.5–15.8 °C for 20 minutes, and then added to the resin in a jacketed filter reactor at 25 °C. The flask was rinsed and transferred out using an additional 5 mL of DMF. The slurry was stirred for 29 hours, then drained and washed with DMF (4 × 300 mL; ~100 mL wash volume should be sufficient), stirring for 5 minutes each time. Another batch of the activated ester prepared as before was added to the reaction mixture and stirred at 25 °C for 19 hours. The resin was drained and washed with DMF (4 × 300 mL; ~100 mL wash volume should be sufficient) and dichloromethane (4 × 300 mL; ~100 mL wash volume should be sufficient), stirring for 4 minutes each time. The PYBOP batch used was determined to be of suboptimal quality; therefore, another batch of activated ester (prepared using a new vial of PYBOP at 1 / 3 the scale of the previous reagent in 50 g DMF) was added to the reaction mixture and stirred at 25°C for 18 hours. The resin was drained and washed with DMF (4 × 300 mL; ~100 mL wash volume should be sufficient) and isopropanol (4 × 300 mL; ~100 mL wash volume should be sufficient), with stirring for 5 minutes each wash. In manufacturing runs, PYBOP should be of high quality, and only a single charge of activated ester should be used for coupling of the intermediate of Formula I. The resin was dried overnight under vacuum at 40°C.

[0265] The cleavage cocktail was prepared as follows: Dithiothreitol (DTT; 12.33 g, 79.93 mmol) and water (17.63 g) were combined in a 5-liter round-bottom flask. TFA (538 g, 4720 mmol) was loaded into a glass pressure vessel and inertized by four pressure purging cycles with nitrogen at 5–10 psig. 60 g of inertized TFA was added to the 5-liter round-bottom flask in 20 g portions at 2-minute intervals, followed by the addition of the remaining TFA over 2-minute intervals. Triisopropylsilane (9.37 g, 59.2 mmol) and dichloromethane (51 g) were added to the 5-liter round-bottom flask, and the mixture was cooled to 15°C.

[0266] To cleave and deprotect the peptide from the resin, dried resin was loaded into a filtration reactor and washed with dichloromethane. The cleavage mixture was added to the resin in the filtration reactor and stirred for 4.5 hours. The reaction solution was leached from the resin and added to a 5-liter round-bottom flask in which the cleavage mixture was prepared. Dichloromethane (206 g) was added to the resin, stirred for 5 minutes, leached, and added to the 5-liter round-bottom flask. The resulting mixture was stirred and cooled at a jacket setpoint of -15°C. MTBE (754 g), which had been inertized by bubbling with nitrogen for 15 minutes and then kept under slow nitrogen purging, was added over 1.5 hours. The slurry was heated to -8°C and stirred for 1 hour, then heated to 0°C and stirred for 1 hour. The slurry was filtered under nitrogen, not allowed to dry completely, and then the solids were washed with MTBE (2 × 155 g) and dried under vacuum to obtain crude tirzepatide (38.7 g), which had a potency of 40.9% as determined by HPLC. During the deprotection of -ivDde, coupling with the acid of formula I, and cleavage from the resin, the sample was extracted from the mixture, totaling 0.34 mmol of the starting resin. Taking this into account and the HPLC titer, the crude yield of tirzepitide in this process was 48%.

[0267] Batch process method

[0268] For comparison, a batch manufacturing process for tirzepatide was described. Fmoc Sieber resin (17 kg, 0.76 mmol / g) was loaded into a reactor. The resin was swollen with DMF, stirred for 2 hours, and then the DMF was filtered off from the resin. The resin was then washed with DMF a total of twice. The Fmoc-protected resin was then deprotected using a 20% PIP / NMP treatment. Sampling was performed after the final PIP / NMP treatment to verify Fmoc removal, which was confirmed by UV analysis to be >99% Fmoc removal. After the final 20% w / w PIP / NMP treatment, the resin bed was washed with DMF multiple times. The peptide backbone was then constructed using the following general conditions for each amino acid coupling and deprotection:

[0269]

[0270] Fmoc deprotection:

[0271] The resin in the peptide reactor was treated with 20% v / v PIP / NMP solution in 3 or 4 charge cycles. Each treatment involved stirring the resin for 30 minutes, followed by filtration to complete the removal of Fmoc protecting groups. After the final 20% v / v PIP / NMP treatment, the resin bed was washed at least six times with a pre-specified DMF charge volume.

[0272] Amino acid activation:

[0273] A pre-prepared 12% w / w Oxyma Pure / NMP solution was loaded into the reactor. The selected Fmoc amino acids were then added. The mixture was stirred at 20 ± 5 °C until the Fmoc amino acids were completely dissolved. The Fmoc-AA / Oxyma Pure / NMP solution was then cooled to 15 ± 3 °C before activation to ensure control of the slightly exothermic activation reaction and to maintain the temperature of the resulting solution within the specified range of 20 ± 5 °C. The amino acid solution was then activated by adding DIC. The activated ester solution was then stirred for 20–30 minutes, and the solution was transferred to a reactor containing the peptide intermediate on the resin.

[0274] Couplet:

[0275] After the pre-activation step, the activated ester solution is transferred to a reactor containing the deprotected peptide on the resin to initiate the coupling reaction. This peptide coupling reaction is stirred at 20 ± 5 °C for at least 4 hours. After the desired stirring time, the resin slurry is sampled to determine coupling completion (IPC). Sampling is repeated at specific intervals as needed until a passing IPC result is obtained. If necessary, a re-coupling operation is performed. When coupling is complete, the contents of the peptide reactor solution are filtered, and the peptide intermediate on the resin is then washed several times with DMF to prepare for the next coupling.

[0276] Ile (12) to Aib (13) coupling:

[0277] The coupling of Fmoc-Ile(12) to Aib(13) was performed using the symmetrical anhydride method, employing 6 equivalents of Fmoc-AA and 3 equivalents of DIC. The activation time for this sequence was extended to 40–60 minutes to ensure the formation of activated symmetrical anhydrides. Extended coupling stirring time (18 hours) was required to achieve the desired reaction completion (<1% uncoupled) as determined by HPLC analysis.

[0278] In the intermediate of the 39-amino acid main chain ( Figure 7 Summary of materials used in the synthesis of ) :

[0279] For amino acid coupling, an average of 3.15 piperidine loads were used per cycle at 9 mL / g resin, with a density of 0.93 g / mL and an average of 26.4 g / g resin. After deprotection, an average of 7.2 DMF washes were performed at 9 mL / g resin, with a density of 0.945 g / mL and an average of 61.2 g / g resin per cycle. For amino acid coupling, a coupling solution with an average density of approximately 1 g / mL was used per cycle, with an average of 7.25 g / g resin per cycle. After coupling, an average of 5 DMF washes were performed at 9 mL / g resin, with a density of 0.945 g / mL and an average of 42.5 g / g resin per cycle.

[0280] Each cycle uses an average of 137 grams of material per gram of resin. This is for the production of the 39-amino acid main chain intermediate (…). Figure 7 The experiment was conducted over 39 cycles, using 5356 g of material / g of resin. Since the molar capacity of Sieber resin is 0.7 mmol / g, the total mass of material used per millimole of resin (including the mass of the resin itself) is approximately 7650 g / mmol.

[0281] Lys-ivDde(20) deprotection, coupling to formula I, resin pyrolysis, crude product separation:

[0282] The remaining steps for deprotecting Lys-ivDde, coupling with acid I, pyrolysis of the resin, and separation of crude tirzepatide were performed substantially as described for the preparation of the tandem reactor. Overall, 45.39 kg of crude tirzepatide was produced with a purity of 45 wt% and 64% HPLC area %. The contained yield based on Sieber resin was 47%.

[0283] Method Comparison

[0284] Similar crude yields (47% and 48%, respectively) were obtained through batch and tandem reactor production of tirzepatide. For the tandem reactor method, 5.42 kg of total material (solvent + reagent + resin) was used per millimole of starting resin to produce the 39-mer main-chain intermediate. Figure 7 In the batch process, 7.65 kg of total material is used per millimole of starting resin.

[0285] Example: Fragmentation preparation with DMF recycling and reduced reagent equivalents

[0286] An improved setup of three reactors in series was used to synthesize peptides containing the first 10 amino acid residues of the tirzepatide backbone, namely GPSSGAPPPS-NH-resin, but each S amino acid was protected by a tert-butyl group.

[0287] The device is set up with Figure 3 The same as shown, only with minor adjustments, in which Figure 3 The peristaltic pump (435) is replaced by a pressure transmission system. This system evacuates the transfer can instead of pumping out the solvent, then opens the feed valve from the desired reactor (RB1, RB2, or RB3). Pressure sensors are used to determine when the reactor is completely emptied, as the pressure increases rapidly once nitrogen, instead of solvent, is introduced into the can. The transfer can is then pressurized, and the valve to the desired reactor (RB2, RB3, or waste / recycle) is opened. This technique allows for faster and more complete emptying of the reactor, resulting in less residual solvent after emptying, which improves resin washing. Another difference is the addition of an online GC (gas chromatograph) to measure piperidine to ensure adequate washing, and a DMF recirculation bottle.

[0288] Regarding Figure 3The system was similar to that of the other reactors, with three reactors set up in series. Effluent from the first reactor was transferred to the second reactor, from the second reactor to the third reactor, and from the third reactor to waste. However, only the washing operation was carried out in series. The experiment also used improved conditions for both Fmoc deprotection and amino acid coupling reactions. The new reaction conditions eliminated most of the excess reagent and made the concept of carrying out deprotection and coupling reactions in series have a minimal benefit. For Fmoc deprotection, only a single 20% piperidine / DMF charge was used. The volume of this charge was reduced to approximately four volumes (based on the initial dry resin charge). At this point, piperidine waste was reduced by approximately 80% compared to the original method, which used more than three independent nine-volume charges in a single reactor. The potential piperidine savings from carrying out this operation in series were deemed not worthwhile because potential problems could arise from the accumulation of deprotection byproducts (piperidine-dibenzo[a]-enene adducts) in the third reactor. Therefore, each reactor was operated independently for the Fmoc deprotection reaction.

[0289] To further minimize PMI, the system was modified to include a DMF recycling vessel. (PMI, or Process Mass Intensity, is defined as the total mass of material used to prepare a specified amount of product; therefore, 2000 grams of material used to prepare 1 gram of product has a PMI of 2000.) The recycling vessel is used to collect the latter half of the wash solution after Fmoc deprotection and is located downstream of the third reactor, as all washes are performed in series. For example, the recycling vessel collects the last five of ten washes. The collected DMF has a piperidine concentration of approximately 1%. This is significantly lower than the piperidine concentration in the reactor after the deprotection reaction, and the collected DMF is used for the first half of subsequent wash cycles. This allows for better utilization of the DMF and increases the average piperidine concentration in the waste stream. Additional modifications were made to the washing process due to the need to wash the (as described above) online LCMS equipment to avoid sample entrainment. The LCMS equipment wash produces approximately 80 ml of DMF in each of the three reactors. A quick sprayball wash of approximately 50 ml of fresh DMF is then performed on each reactor to ensure any resin on the walls is flushed into the resin bed. The online LCMS and spray ball scrubbing are integrated into the series reactor scrubbing program, in which all DMF used is transferred through the series reactors and included in the final PMI calculation.

[0290] Previously in Figure 3In this process, peristaltic pumps are used to transfer material from one reactor to the next. Sometimes, the peristaltic pumps do not completely empty the free solvent from the reactor. This reduces washing efficiency and increases PMI (Potential Intake). Therefore, a change is made to use pressure transfer to better empty all liquids from the reactor. A transfer container is added and connected to all reactors, waste, and DMF recycling containers. An automated sequence is used to transfer material from the reactor to the transfer container. A vacuum is created on the transfer container, and the valve between the bottom of the transfer tanks is opened. When the pressure in the transfer container reaches a predetermined setpoint, the valve closes. The transfer container is then pressurized, the valve to the top of the target container is opened, and transfer occurs until the pressure in the transfer container drops below the set value.

[0291] Online analysis of the washing solution was also added. An online GC was set up in the waste line to monitor residual piperidine in the washing solution. This procedure was set up to analyze the last three wash solutions eluted from the third reactor. Additionally, online LCMS was used to sample the washing solution to determine residual amino acids during post-coupling washing. To prevent yield reduction, resin settling was allowed before triggering the washing sample. This prevented resin from being sampled because the immersion tube would be in the liquid layer above the resin.

[0292] Accurate PMI calculations were obtained for each cycle. For this specific experiment, all reaction monitoring samples were taken from reactor 3, as it was expected to have the slowest reaction due to the highest amount of residual material, given its lower washing efficiency. Once the reaction in reactor 3 was complete, it was assumed that all three reactors were complete. This also helps with the entrainment problem in online LCMS, as each sample is only slightly affected by entrainment. Under the assumption of 5% entrainment, a 100% complete coupling reaction might only be measured as 95% complete due to 5% of the previous deprotection reaction samples. If each of the three reactors was sampled sequentially, this 5% error could exist in each sample, and the reaction might still appear incomplete after 3 hours. If only one reactor was sampled, assuming complete conversion, the first sample had a conversion of 95%, but the second sample measured 99.75% conversion and the reaction could be stopped after only 2 hours. Experimental history shows that reactions exceeding the necessary time are associated with compromised quality, especially in Fmoc deprotection reactions. The risks of sampling only one reactor are considered negligible compared to the potential quality improvements that could result from stopping the reaction when it is complete (without excessive additional stirring time).

[0293] The goal of this experiment was to reduce the piperidine concentration to 2000 ppm using 10 washing cycles. The results of the washing after piperidine deprotection are shown in Table 10. The model was used to predict the amount of wash charge required to obtain approximately 2000 ppm piperidine after 10 washing and sample cart cleaning cycles. This simulation utilized previous data from resin swelling and piperidine adsorption experiments. Some samples were missed due to equipment malfunction, but the target of 2000 ppm was generally achieved, with the sole exception of the last cycle.

[0294] Table 10: Piperidine Washing Results

[0295] Synthesis cycle # AA deprotection washing# Piperidine concentration (ppm) 1 Sieber resin 10 1804 1 Sieber resin Sample cart cleaning 950 2 Ser 39 10 2100 2 Ser 39 Sample cart cleaning 1003 3 Pro 38 10 3605 3 Pro 38 Sample cart cleaning 1723 4 Pro 37 10 5215 4 Pro 37 Sample cart cleaning 2013 5 Pro 36 9 4164 5 Pro 36 10 4132 5 Pro 36 Sample cart cleaning 1715 6 Ala 35 9 3393 6 Ala 35 10 1605 7 Gly 34 9 3473 7 Gly 34 10 1452 8 Ser 33 9 3871 9 Ser 32 9 4069 9 Ser 32 Sample cart cleaning 1927 10 Pro 31 9 4982 10 Pro 31 10 4232 10 Pro 31 Sample cart cleaning 3048

[0296] Following the coupling reaction, the system was washed three times consecutively with the wash charge. The same sample cart cleaning procedure was then used thereafter. No good method for quantifying residual AA has been developed; therefore, increasing amounts of DMF were used with each wash charge as peptide growth and solvent retention increased.

[0297] Reaction monitoring was performed on a third reactor connected in series. An online LC-MS system was used as in previous experiments. Deprotection results are presented in Table 11. Given that this quantitative method is prone to underreporting and that sample analysis occurred one hour later than the actual reaction, a conversion rate of 98% was expected before stopping the reaction and continuing. All deprotections reached the threshold for continuation two hours after processing the sample. The processing time was just over one hour.

[0298] Table 11: Results of Deprotection Reaction

[0299]

[0300] Similar monitoring was performed on the coupling reactions. The results are presented in Table 12. All couplings except for Gly 30 were stopped no later than two hours after the results for the samples were obtained. The general theory for the low conversion of Gly 30 coupling is the presence of high levels of residual piperidine after washing. The Gly 30 reaction was recoupled, which rapidly brought the coupling conversion to 99.2%, and the reaction was then stopped.

[0301] Table 12: Results of Coupling Reaction

[0302]

[0303] The main focus of this experiment was to prepare high-quality materials with minimal amounts of raw materials. The amount of material used in each deprotection cycle is listed in Table 13. The amount of material per cycle is given in g / mmol. Since the number of millimoles of peptide decreases in each cycle, adding up the total amount of material used for 10 cycles and dividing by the final or initial number of millimoles does not yield an accurate figure.

[0304] Table 13: Materials Used for Deprotection

[0305]

[0306] The amount of DMF used for washing after deprotection is shown in Table 14. Table 15 lists the molar equivalents of the coupling agent used for each cycle for each of the three reactors. The total material used for each coupling cycle is given in Table 16. Table 17 gives the amount of DMF used for washing after coupling.

[0307] Table 14: Materials used for washing after deprotection

[0308]

[0309] Table 15: Molar equivalents of coupling reagents used

[0310]

[0311] Table 16: Total Coupling Reagents Used

[0312]

[0313] Table 17: DMF dosage for post-coupling washing

[0314]

[0315] Data from each step was used to generate a summary comparing raw material usage (by compound and total) with the current batch manufacturing process. Table 18 shows a 37% reduction in coupling reagent usage and an 84% reduction in piperidine usage. DMF used for washing constitutes the vast majority of materials used in this process, well over 90% of the total mass consumption; reducing DMF consumption was a primary objective of this experiment. DMF usage was reduced by 79% compared to the first ten cycles of the tirzepatide batch manufacturing process. The experimental demonstration of this new process technology used a total of 357 g / mmol of peptide. In contrast, the current batch manufacturing process uses 1690 g / mmol. This represents a total reduction of 79%.

[0316] Table 18: Total materials used in 10mer SPPS in tandem and batch processes

[0317]

[0318] After confirming a reduction in material consumption of nearly 80%, the final part of the experiment was to demonstrate the high quality of the resulting material. The material from all three reactors underwent soft cleavage of the peptide from the resin, while retaining all protecting groups on the AA R groups. The HPLC analysis shown in Table 19 demonstrates the high quality achieved after passing through all three reactors. Somewhat surprisingly, there was absolutely no correlation between quality and reactor type. The hypothesis is that the purity decreased slightly after passing through the three reactors because the third reactor, in series, did not wash as much as the first reactor. This finding demonstrates that the washing strategy used exceeded a sufficient level. Theoretically, the washing targets for residual piperidine and AA could be increased, which would allow for further reductions in material consumption.

[0319] Table 19: HPLC purity data for each reactor

[0320]

[0321] Example: Construction of Terzepatide tetramer by SPPS in DMF solvent

[0322] The following Tirzepatide tetramer contains 4 amino acids:

[0323]

[0324] This tetramer was synthesized using linear solid-phase peptide synthesis (SPPS), achieved via the loading method and tandem reactor method described below. Table 20 lists the sequence of the four amino acids used to synthesize the tirzepatide tetramer backbone. It should be noted that the loading method was first used to load the first amino acid of the tetramer intermediate, glycine, onto a 2-chlorotriphenylmethylchloro (CTC) resin solid support, as shown below. (The CTC resin solid is shown as a circle below).

[0325]

[0326] Table 20. Sequence of the four amino acids used to synthesize the Tirzepatide tetramer via SPPS

[0327] amino acid (AA) addition order AA position on peptide AA Name AA for coupling steps 1 4 glycine Fmoc-Gly-OH 2 3 glutamic acid Fmoc-Glu(Bu)-OH 3 2 2-Aminoisobutyric acid Fmoc-Aib-OH 4 1 Tyrosine Boc-Tyr(tBu)-OH

[0328] The remaining three amino acids were constructed using a tandem reactor method. The α-nitrogen groups of glycine, glutamic acid, and 2-aminoisobutyric acid were protected by a 9-fluorenylmethoxycarbonyl group (Fmoc), while the α-nitrogen group of tyrosine was protected by a tert-butyloxycarbonyl group (Boc). The oxygen groups of glutamic acid and tyrosine were protected by a tert-butyl group (tBu).

[0329] Loading method

[0330] Preparation of Fmoc-Gly-OH coupling solution: In a 5000 mL sample vial, Fmoc-Gly-OH (142.7 g, 480.0 mmol) was dissolved in DMF to a volume of 1.8 L. Then, N-ethyl-N-isopropyl-propyl-2-amine (309 g, 2390.9 mmol) was added and the contents were stirred for 5 minutes.

[0331] Resin Coupling

[0332] Add CTC resin (380 g, 610 mmol, 1.6 mmol / g) to a 10 L glass large-scale peptide synthesizer. Add DMF (2400 mL) and stir for 15 minutes. Leach and discard the remaining solution. Wash the resin with DMF (2400 mL) for 15 minutes, stirring twice. Add additional N-ethyl-N-isopropyl-propyl-2-amine (62 g, 479.73 mmol) and stir at ambient temperature for a total of 18 hours. After 18 hours, leach the solution and wash the resin five times with DMF (1800 mL), mixing for 5 minutes each time.

[0333] Resin sealing

[0334] DMF (900 mL), N-ethyl-N-isopropyl-propyl-2-amine (145.8 g, 1128 mmol), and methanol (191 g, 5960.9 mmol) were added to a flask and stirred for 2 minutes at room temperature. The solution was added to the resin and stirred for 60 minutes, after which the solution was leached. The resin was washed five times with DMF (1800 mL), stirring for 5 minutes each time. The resin was then transferred to a drying tray and dried under vacuum at 35 °C to constant weight.

[0335] The dry resin weighed 473.37 g and was found to have a Fmoc-Gly-OH loading of 0.85 mmol / g by NMR.

[0336] Series reactor method

[0337] Raw material preparation

[0338] A 20 vol% solution of piperidine in DMF was prepared as follows: Piperidine (2.0 L) was diluted to a volume of 10.0 L by adding DMF to obtain a 20 vol% solution.

[0339] A 1.25 mol / kg solution of oxyma in DMF was prepared as follows: Ethyl cyanoacetate (oxyma, 195.69 g) was dissolved in DMF (905.91 g) until a 1.25 mol / kg solution was obtained, and then nitrogen gas was bubbled through the solution at 2 SCFH.

[0340] A 1.25 mol / kg solution of DIC in DMF was prepared as follows: N,N'-5-diisopropylcarbodiimide (191.16 g) was dissolved in DMF (1020.60 g) until a 1.25 mol / kg solution was obtained, and then nitrogen gas was bubbled through the solution at 2 SCFH.

[0341] The following is a method for preparing a 0.40 mol / kg solution of glutamic acid in DMF: Fmoc-Glu( t Bu)-OH (162.85 g) was dissolved in DMF (755.15 g), shaken to dissolve, and then nitrogen gas was bubbled through the solution at 2 SCFH.

[0342] A 0.40 mol / kg solution of Fmoc-Aib-OH in DMF was prepared as follows: Fmoc-Aib-OH (119.50 g) was dissolved in DMF (798.51 g), shaken to dissolve, and then nitrogen gas was bubbled through the solution at 2 SCFH.

[0343] A 0.40 mol / kg solution of Boc-Tyr(tBu)-OH in DMF was prepared as follows: 123.89 g of Boc-Tyr(tBu)-OH was dissolved in 794.11 g of DMF, shaken to dissolve, and then nitrogen gas was bubbled through the solution at 2 SCFH.

[0344] The reaction system was prepared as follows: CTC resin (108 g, 0.85 mmol / g, 91.8 mmol) was added and divided equally between reactors “RB1”, “RB2”, and “RB3”. 350 mL of DMF was added to each reactor and the mixture was stirred at room temperature for 2 hours to allow the resin to swell.

[0345] General Program A - Fmoc Deprotection and DMF Washing Process:Fmoc deprotection was performed in parallel in all three reactors, RB1, RB2, and RB3. More specifically, piperidine solution (20 vol% in DMF, 202.4 g) was added to RB1, and the piperidine transfer line was flushed with 5 ml of DMF and stirring was initiated. Similarly, 205.4 g and 205.2 g of 20 vol% piperidine solution were added to RB2 and RB3, respectively, and the piperidine lines were flushed. The three reactors were then simultaneously stirred at 20 °C for 120 min to perform deprotection in parallel. The piperidine solution loadings used for the overall TZP tetramer construction are summarized in Table 21. The deprotection reaction was monitored using online LC-MS supported by a sampling cart. The deprotection conversion could not be quantitatively determined because the mass spectra of the deprotected tetramer intermediate were not tabulated due to their low molecular weight. However, qualitative verification of deprotection completion ensured that the extracted ion peak of the Fmoc-protected tetramer was zero.

[0346] After deprotection, the solution is drained from all three reactors into a waste tank using a pressure transfer system. It should be noted that the DMF washing after deprotection is performed using a tandem reactor approach, i.e., the DMF washing solvent is first loaded into RB1, then transferred to RB2 and finally to RB3. To remove residual piperidine from the resin, 10 DMF washing cycles are applied using the following detailed procedure. For the first 5 DMF washes, recirculated DMF from the DMF recirculation vessel is added to RB1 in equal portions by solvent mass and stirred (i.e., the mass of DMF in the recirculation vessel is divided equally into 5 washing cycles and loaded into RB1). After the DMF recirculation vessel is emptied and the first five DMF recirculation washes are completed, fresh DMF from the DMF solvent vessel is loaded for the remaining 5 wash cycles. The used DMF from the first 5 wash cycles is discarded into the waste tank, while the used DMF solvent from the remaining 5 wash cycles is collected in the DMF recirculation tank for reuse in the next amino acid construction. A stirring time of 5 minutes is applied throughout the washing cycle. After 10 washing cycles, an additional sample cart cleaning cycle was applied, in which 100 mL of fresh DMF was loaded into each of the three reactors. The DMF from RB1 and RB2 was then transferred to RB3, where it was finally collected in the DMF solvent recycling vessel. The materials used for washing the DMF after deprotection are summarized in Table 22. The piperidine concentration of the DMF solution discharged from the last three washing cycles was measured using online GC, and is summarized in Table 23. The final residual piperidine concentration was controlled below 500 ppm throughout the synthesis.

[0347] Table 21 Materials for Deprotection

[0348]

[0349] Table 22 Material usage for washing after deprotection.

[0350]

[0351] Table 23 Residual piperidine concentration after deprotection washing

[0352] Synthesis cycle AA deprotection washing# PPM 1 Gly 4 9th No data 1 Gly 4 10th No data 1 Gly 4 Sample cleaning and washing 369.1 2 Glu 3 9th 535.7 2 Glu 3 10th 506.1 2 Glu 3 Sample cleaning and washing 337.8 3 Aib 2 9th 555.8 3 Aib 2 10th 474.2 3 Aib 2 Sample cleaning and washing 409.7

[0353] General Procedure B – Amino Acid Activation and Coupling Process: A glutamate solution (0.4 mol / kg, 401.2 g), an oxyma solution (1.25 mol / kg, 128.5 g), and a DIC solution (1.25 mol / kg, 141.2 g) were added to jacketed reactor RA. The solution was stirred at 20°C for 30 min to form an activated glutamate solution, which was then added to RB1 (225.4 g), RB2 (225.4 g), and RB3 (227.4 g). Reactors RB1, RB2, and RB3 were stirred at 20°C for 8 h, except for tyrosine coupling (where an 18-hour coupling time was used). After the coupling time was reached, the solution was drained from all three reactors into a waste tank using pressure transfer. At the end of the reaction, coupling was confirmed by a Kaiser test. The amounts of materials used for coupling are summarized in Table 24, which details the equivalent ratios used throughout the construction process.

[0354] For the three cycles, fresh DMF washing reactors RA, RB1, RB2, and RB3 are used in a similar series reactor configuration to General Procedure A after coupling. More specifically, fresh DMF solvent is added to RA via a spray valve, and then introduced in series into RB1, RB2, and RB3. It should be noted that the sample cart cleaning procedure is performed in a manner similar to General Procedure A. Subsequently, all used DMF from the three DMF washing cycles and an additional sample cart cleaning cycle is disposed of in a waste container. The materials used for DMF washing after coupling are summarized in Table 25.

[0355] Table 24 Material Usage for Coupling

[0356]

[0357] Table 25 Material Usage for Washing After Coupling

[0358]

[0359] TZP tetramer purity analysis method

[0360] Following Boc-Tyr 1 coupling, the resin was transferred from all three reactors to a drying tray, deswollen by washing with dichloromethane, and dried. Tetramer samples were soft-cleaved using tBu, with the Boc protecting group retained on the peptide, and their purity was analyzed using the following method: For every 500 mg of peptide on the resin, 10 mL of 30% hexafluoro-2-propanol / dichloromethane (v / v) was added to a scintillation flask. The mixture was stirred on a rotary mixer for 2 h. The resin cake was filtered and washed with 10 mL of dichloromethane. The mixture was concentrated to an oil by rotary evaporation. This tetramer oil was further diluted with 50% acetonitrile / water (v / v) for UPLC-MS analysis. The resulting TZP tetramers from RB1, RB2, and RB3 had purities of 99.23%, 99.58%, and 99.39%, respectively.

[0361] Batch process method for TZP tetramer synthesis

[0362] To compare with the series reactor technology, the TZP tetramer batch process at 600 mmol is described below. It should be noted that a single batch reactor was used for this batch process instead of the three series batch reactors used in the new technology; the material amounts are summarized in Table 26.

[0363] Fmoc-Gly-OH loading

[0364] CTC resin (500 g) was loaded into the reactor. The resin was swollen with DMF and stirred. Fmoc-Gly-OH was added to the reactor along with DMF / DCM and loaded for 2 hours. The solution was leached and washed with DMF. DMF / DIPEA / MeOH was added to the reactor twice and leached for capping, each time stirred for 20 minutes. The Gly-loaded resin was further washed with DMF. The loading amount was determined to be 1.2 mmol / g.

[0365] Fmoc deprotection

[0366] Fmoc deprotection was performed using 20% ​​piperidine / DMF with two stirring cycles. For each stirring, a reaction time of 80 minutes was used, and the deprotecting agent was leached and filtered. After the deprotection reaction was complete, the mixture was washed eight times with 6.5 volumes of DMF, stirring for 5 minutes each time. Deprotection was confirmed to be complete using a tetrachloroquinone test.

[0367] Amino acid activation

[0368] An oxyma solution (2 eq., 1.25 mol / kg) was added to the pre-activated reactor, followed by the addition of amino acids (2 equivalents) along with additional DMF solvent to dilute the amino acid concentration to 0.4 mol / kg. Then, a DIC solution (2.2 eq., 1.25 mol / kg) was added to the reactor to initiate activation. An activation time of 2 hours was applied to Glu, while stirring for 15 minutes was applied to Aib and Tyr. The activation process was maintained at 20°C.

[0369] Couplet

[0370] The activated ester solution was transferred to a reactor containing the deprotected peptides on the resin. Stirring was applied for 8 hours for Glu and Aib, while Tyr coupling required 18 hours. A Kaiser assay was used to confirm the completion of the reaction. Once coupling was complete, the coupling solution was leached and washed with 3 x 6.5 vol. DMF to remove any remaining activated esters.

[0371] Purity Analysis

[0372] A similar soft cleavage analysis method was used in conjunction with UPLC-MS to determine TZP tetramer samples from batch processes. The purity was determined to be 99.6%.

[0373] Table 26. Material usage for the manufacturing batch process used in the synthesis of TZP tetramer at a scale of 600 mmol.

[0374]

[0375] Method Comparison

[0376] Similar TZP tetramer purities (99.3% and 99.6%, respectively) were obtained using both the tandem reactor method and the batch process. For the tandem reactor method, 118.9 g of total material (solvents, reagents, excluding resin) was used per millimole of starting resin to produce TZP tetramer. In the batch process, 218.8 g of total material (solvents, reagents, excluding resin) was applied per millimole of starting resin. Overall, the novel tandem reactor method reduced PMI by 45.7% compared to the conventional batch process.

[0377] The embodiment is through N Solid-phase peptide synthesis (SPPS) in a green alternative solvent of butylpyrrolidone / furan Construct tirzepatide tetramer

[0378] Significant amounts of toxic solvents, such as dimethylformamide, are used in conventional solid-phase peptide synthesis. NMethyl-2-pyrrolidone, dimethylacetamide, and dichloromethane pose challenges to industrial hygiene and environmental protection. Therefore, there is interest in developing greener alternative solvents with PMI reduction benefits for tandem reactor processes in next-generation SPPS technologies. The NBP / furan (particularly tetrahydrofuran and 2-methyltetrahydrofuran) dual system has been selected from extensive pre-screening studies as an alternative green solvent due to its excellent polystyrene-based resin swelling properties, coupling agent solubility, and high coupling and deprotection reaction performance. To make a back-to-back comparison with the TZP (terzepatide) tetramer synthesis using DMF solvent in the previous example, the tirzepatide tetramer was synthesized using the NBP / THF green alternative solvent system:

[0379] .

[0380] This tetramer was constructed using the tandem reactor method described below. The amino acid sequence is shown in Table 27, where 9-fluorenylmethoxycarbonyl (Fmoc) protects the α-nitrogen of glycine, glutamic acid, and 2-aminoisobutyric acid, while tert-butyloxycarbonyl (Boc) and tert-butyl (Fmoc) protect the α-nitrogen of glycine, glutamic acid, and 2-aminoisobutyric acid. t Bu) is used to protect the α-nitrogen of tyrosine and the oxygen of glutamate, respectively. It should be noted that the Fmoc-Gly-OH-loaded CTC resin (loading amount 0.85 mmol / g) prepared in the TZP tetramer synthesis in the DMF solvent examples was used in the NBP / THF green solvent examples. Detailed loading methods can be found in the foregoing examples. The following shows the construction of the remaining three amino acids using a similar tandem reactor method on CTC resin in the NBP / THF solvent system.

[0381]

[0382] Table 27 The sequence of the four amino acids used to synthesize the Tirzepatide tetramer via SPPS

[0383] amino acid (AA) addition order AA position on peptide AA Name AA for coupling steps 1 4 glycine Fmoc-Gly-OH 2 3 glutamic acid Fmoc-Glu(Bu)-OH 3 2 2-Aminoisobutyric acid Fmoc-Aib-OH 4 1 Tyrosine Boc-Tyr(tBu)-OH

[0384] Tandem reactor method using NBP / furan as an alternative solvent

[0385] Raw material preparation

[0386] A 20 vol% solution of piperidine in NBP / THF was prepared as follows: Piperidine (2.0 L) was diluted to a volume of 10.0 L by adding NBP / THF (1.5:1 v:v) to obtain a 20 vol% solution.

[0387] A 0.72 mol / kg solution of oxyma in NBP / THF (2:1, v:v) was prepared as follows: Ethyl cyanoacetate (oxyma, 176.1 g) was dissolved in NBP:THF (2:1, v:v) (1545.1 g) to obtain a 0.72 mol / kg solution, and then nitrogen gas was bubbled through the solution at 2 SCFH.

[0388] A 0.72 mol / kg solution of DIC in NBP / THF (2:1, v:v) was prepared as follows: N,N'-5-diisopropylcarbodiimide (172.0 g) was dissolved in NBP / THF (2:1, v:v) (1721.3 g) to obtain a 0.72 mol / kg solution, and then nitrogen gas was bubbled through the solution at 2 SCFH.

[0389] The following is a sample preparation of a 0.35 mol / kg solution of glutamic acid in NBP / THF (2:1, v:v): Fmoc-Glu( t Bu)-OH (175.8 g) was dissolved in NBP / THF (2:1, v:v) (1004.5 g), shaken to dissolve, and then nitrogen gas was bubbled through the solution with 2 SCFH.

[0390] The following method was used to prepare a 0.35 mol / kg solution of Fmoc-Aib-OH in NBP / THF (2:1, v:v): Fmoc-Aib-OH (139.4 g) was dissolved in NBP / THF (2:1, v:v) (1045.9 g), shaken to dissolve, and then nitrogen gas was bubbled through the solution at 2 SCFH.

[0391] A 0.45 mol / kg solution of Boc-Tyr(tBu)-OH in NBP / THF (2:1, v:v) was prepared as follows: 139.4 g of Boc-Tyr(tBu)-OH was dissolved in 1040.9 g of NBP / THF (2:1, v:v), shaken to dissolve, and then nitrogen gas was bubbled through the solution at 2 SCFH.

[0392] The reaction system was prepared as follows: CTC resin (108 g, 0.85 mmol / g, 91.8 mmol) was added and divided equally between reactors “RB1”, “RB2”, and “RB3”. 350 mL of NBP / THF (1.5:1, v:v) was added to each reactor and the mixture was stirred at 20°C for 1 hour at room temperature to allow the resin to swell.

[0393] General Program A - Fmoc Deprotection and NBP / THF Washing Process:Fmoc deprotection was performed in parallel for specific time amounts in all three reactors RB1, RB2, and RB3 at 30°C in a 20% piperidine solution in NBP / THF (1.5:1, v:v). The solution was then drained into a waste tank and loaded with additional piperidine solution for deprotection stirring until the reaction was complete. More specifically, for the first deprotection stirring using Fmoc-Gly-OH as an example, piperidine solution (20% by volume in NBP / THF (1.5:1, v:v), 209 g) was added to RB1 and the piperidine transfer line was flushed with 5 ml of NBP / THF (1.5:1, v:v) solvent mixture and stirring was initiated. In a similar manner, 208.8 g and 205 g of 20% piperidine solution were added to RB2 and RB3, respectively, and the piperidine lines were flushed. The three reactors were then stirred at 30°C for 45 minutes for parallel deprotection, after which the solution was drained. Online LC-MS sampling and qualitative verification were performed to confirm the completion of the reaction by verifying that the extracted ion peak of the Fmoc-protected tetramer was zero. To completely deprotect Fmoc-Gly-CTC, five additional parallel deprotection stirrings were applied to RB 1 / 2 / 3. A similar deprotection stirring strategy was used for Glu 3 and Aib 4, with two deprotection stirrings performed. Table 28 summarizes the piperidine solution loadings used for the entire TZP tetramer construction.

[0394] An NBP / THF (1.5:1, v:v) solvent mixture was used for post-reaction washing at 30°C, employing a similar tandem reactor and solvent recycling strategy. More specifically, RB1 / 2 / 3 were washed with 10 NBP / THF washing cycles throughout the TZP tetramer construction. The used NBP / THF solvent in the recycling vessel was divided into five equal portions and sequentially added to RB1 / 2 / 3 for the first five washing cycles, with stirring for 5 minutes each. All used NBP / THF solvent from the first five cycles was discarded into a waste container after use. For the remaining five washes, fresh NBP / THF was used for the tandem washing of RB1 / 2 / 3 and collected in the NBP / THF recycling vessel for use in the next amino acid deprotection post-wash. Finally, as a sample cart cleaning cycle, NBP / THF was loaded into each of the three reactors in 100 mL portions and further collected in the recycling vessel. Table 29 summarizes the mass balance used for washing after deprotection, and Table 30 details the residual piperidine concentration throughout the synthesis, which is below 600 PPM for all cycles.

[0395] Table 28 Materials for removing protection at 30°C

[0396]

[0397] Table 29 Material Usage for Washing After Deprotection

[0398]

[0399] Table 30 Residual piperidine concentration after washing following deprotection

[0400] Synthesis cycle AA deprotection washing# PPM 1 Gly 4 9th Unobtainable 1 Gly 4 10th Unobtainable 1 Gly 4 Sample cleaning and washing 416 2 Glu 3 9th 499 2 Glu 3 10th No data 2 Glu 3 Sample cleaning and washing 348 3 Aib 2 9th 593 3 Aib 2 10th 506 3 Aib 2 Sample cleaning and washing Unobtainable

[0401] General Procedure B – Amino Acid Activation and Coupling Processes in NBP / THF: For activation and coupling reactions, the NBP / THF volume ratio was adjusted to 2:1 to achieve better reagent solubility. Using Fmoc-Glu ( t Taking Bu-OH coupling as an example, glutamic acid solution (0.35 mol / kg, 786.4 g), oxyma solution (0.72 mol / kg, 382.4 g), and DIC solution (0.72 mol / kg, 420.6 g) were added to jacketed reactor RA and stirred at 30°C for 70 min to form activated glutamic acid solution, which was then transferred to RB1 (531.9 g), RB2 (529.7 g), and RB3 (533.9 g), respectively. Reactors RB1, RB2, and RB3 were stirred at 30°C for 8 hours, after which the solution was drained from all three reactors into a waste tank. At the end of the reaction, the completion of coupling was checked by Kaiser test. Since Glu coupling was not completed after the first coupling step, Fmoc-Glu (1.5 equivalence ratio) was further added. t The Bu-OH-activated solution was used for an additional 19 hours of coupling until the Kaiser test was passed. However, the remaining Aib 2 and Tyr 1 couplings were performed in a single coupling step, with stirring times of 8 hours and 18 hours, respectively. Table 31 summarizes the detailed material usage during the coupling process.

[0402] For post-coupling washing using NBP / THF (1.5:1, v:v) solvent, three wash cycles were employed to rinse RA, RB1, RB2, and RB3. More specifically, fresh NBP / THF solvent was loaded into RA via a spray valve, and then introduced into RB1, RB2, and RB3 three times in series. A sample cart cleaning procedure similar to that performed after deprotection washing was followed. All used post-coupling solvent was discarded into a waste container. The materials used for post-coupling NBP / THF washing are summarized in Table 32.

[0403] Table 31 Material Usage for Coupling

[0404]

[0405] Table 32 Material Usage for Washing After Coupling

[0406]

[0407] Results Summary

[0408] Following Boc-Tyr 1 coupling, the resin was washed with dichloromethane and dried. The sample was then soft-cleaved and separated using 30% hexafluoro-2-propanol / dichloromethane (v / v) for UPLC-MS analysis. The resulting TZP tetramers from RB1, RB2, and RB3 had purities of 92.19%, 94.64%, and 98.04%, respectively. The major impurity was glutamic acid addition. This is primarily attributed to the double glutamic acid coupling strategy employed in this example. It should be noted that a longer resin swelling time (>8 hours) prior to Fmoc-Gly-CTC deprotection alleviates the challenges of Gly 4 deprotection and Glu 3 coupling. For example, 99.6% TZP tetramer purity was obtained by 12 hours of resin swelling using NBP / 2-MeTHF (1.5:1, v:v) green solvent at a 1 mmol scale. Therefore, significant improvements in peptide purity can be achieved using NBP / furan green solvent and tandem reactor technology under more optimized process conditions. For the tandem reactor process using the NBP / THF green solvent, 252.71 g of total material (solvent, reagents, excluding resin) is used per millimole of starting resin to produce TZP tetramer. The conventional batch process using DMF uses 218.8 g / mmol, while the tandem reactor process using DMF uses 118.9 mmol. The NBP / THF procedure is new and unoptimized, using more total solvent than the DMF procedure. However, for any solvent system, the tandem reactor still uses less solvent than a single batch reactor.

Claims

1. A method for coupling an amino acid "X" to a protected N-group attached to a peptide synthesis resin, comprising: obtaining a first reactor and a second reactor, each of the first reactor and the second reactor containing an amount of a protected N-group attached to a peptide synthesis resin; adding a first amount of a deprotection reagent to the first reactor; removing the first amount of the deprotection reagent from the first reactor; adding the first amount of the deprotection reagent to the second reactor; adding a second amount of the deprotection reagent to the first reactor; removing the first amount of the deprotection reagent from the second reactor; removing the second amount of the deprotection reagent from the first reactor; adding the second amount of the deprotection reagent to the second reactor; removing the second amount of the deprotection reagent from the second reactor; washing the peptide synthesis resin in the first and second reactors with a solvent; adding a first amount of an activated ester of the amino acid "X" to the first reactor; removing the first amount of the activated ester of the amino acid "X" from the first reactor; adding the first amount of the activated ester of the amino acid "X" to the second reactor, adding a second amount of the activated ester of the amino acid "X" to the first reactor; removing the first amount of the activated ester of the amino acid "X" from the second reactor; removing the second amount of the activated ester of the amino acid "X" from the first reactor; adding the second amount of the activated ester of the amino acid "X" to the second reactor; removing the second amount of the activated ester of the amino acid "X" from the second reactor; and washing the peptide synthesis resin in the first and second reactors with a solvent.

2. The method of claim 1, wherein the amino acid "X" present in the first and second amounts of the activated ester of the amino acid "X" itself has a protected N-group.

3. The method of claim 1, further comprising: adding a first amount of a deprotection reagent to a third reactor, wherein such addition is made after removing the first amount of the deprotection reagent from the second reactor, wherein the third reactor contains an amount of a protected N-group attached to a peptide synthesis resin; and adding a second amount of the deprotection reagent to the third reactor, wherein such addition is made after removing the second amount of the deprotection reagent from the second reactor.

4. The method of claim 3, further comprising: adding a first amount of the activated ester of the amino acid "X" to the third reactor, wherein such addition is made after removing the first amount of the activated ester of the amino acid "X" from the second reactor; and adding a second amount of the activated ester of the amino acid "X" to the third reactor, wherein such addition is made after removing the second amount of the activated ester of the amino acid "X" from the second reactor.

5. The method of claim 4, further comprising: adding a third amount of the deprotection reagent to the first reactor, wherein such third amount of the deprotection reagent is added to the first reactor after the second amount of the deprotection reagent has been removed from the first reactor; and transferring the third amount of the deprotection reagent from the first reactor to the second reactor, wherein such transfer is made after removing the second amount of the deprotection reagent from the second reactor; and transferring the third amount of the deprotection reagent from the second reactor to the third reactor; and transferring the third amount of the deprotection reagent from the third reactor to the first reactor. removing a third amount of the deprotecting agent from the third reactor.

6. The method of claim 5, further comprising: adding a third amount of the amino acid "X" activated ester to the first reactor, wherein the adding is performed after removing the second amount of the amino acid "X" activated ester from the first reactor; transferring the third amount of the amino acid "X" activated ester to the second reactor, wherein the transferring is performed after removing the second amount of the amino acid "X" activated ester from the second reactor; transferring the third amount of the amino acid "X" activated ester from the second reactor to the third reactor; and removing the third amount of the amino acid "X" activated ester from the third reactor.

7. The method of claim 6, further comprising: removing a first amount of the amino acid "X" activated ester from the third reactor; and adding the first amount of the amino acid "X" activated ester back to the first reactor.

8. The method of claim 7, further comprising: removing a first amount of the deprotecting agent from the third reactor; and adding the first amount of the deprotecting agent back to the first reactor.

9. The method of claim 1, wherein the washing of the first and second reactors with solvent is performed by adding solvent to the reactors.

10. The method of claim 2, wherein after the second amount of the amino acid "X" activated ester has been removed from the first reactor, the method further comprises: adding a first additional amount of the deprotecting agent to the first reactor; transferring the first additional amount of the deprotecting agent from the first reactor to the second reactor; adding a second additional amount of the deprotecting agent to the first reactor; removing the first additional amount of the deprotecting agent from the second reactor; transferring the second additional amount of the deprotecting agent from the first reactor to the second reactor; removing the second additional amount of the deprotecting agent from the second reactor.

11. The method of claim 10, wherein after the second additional amount of the deprotecting agent is removed from the first reactor, further comprising: adding a first amount of the amino acid "Z" activated ester to the first reactor; transferring the first amount of the amino acid "Z" activated ester to the second reactor; adding a second amount of the amino acid "Z" activated ester to the first reactor; removing the first amount of the amino acid "Z" activated ester from the second reactor; transferring the second amount of the amino acid "Z" activated ester from the first reactor to the second reactor; and removing the second amount of the amino acid "Z" activated ester from the second reactor.

12. The method of claim 11, further comprising: adding a first additional amount of the deprotecting agent to the third reactor, wherein the adding is performed after removing the first additional amount of the deprotecting agent from the second reactor, wherein the third reactor contains a quantity of the protected N-group of the amino acid "X" activated ester; and adding a second additional amount of the deprotecting agent to the third reactor, wherein the adding is performed after removing the second amount of the deprotecting agent from the second reactor.

13. The method of claim 12, further comprising: adding a first amount of the amino acid "Z" activated ester to the third reactor, wherein the adding is performed after removing the first amount of the amino acid "Z" from the second reactor; and removing the first amount of the amino acid "Z" activated ester from the third reactor. ​ ​ ​ adding a second amount of the amino acid "Z" activated ester to the second reactor, wherein this addition is made after removing the second amount of the amino acid "Z" activated ester from the second reactor.

14. The method of claim 1, wherein the peptide synthesis resin in the first and second reactors is a Seiber or Rink resin.

15. The method of claim 1, wherein the peptide synthesis resin in the first and second reactors is a Wang resin or a CTC resin.

16. The method of claim 1, wherein the amount of amino acid X added is between 1.1 and 1.6 equivalents.

17. A method of coupling an amino acid "X" to a protected N-group attached to a peptide synthesis resin present in a first reactor and a second reactor, comprising: adding a first amount of a deprotection reagent to the first reactor; removing the first amount of the deprotection reagent from the first reactor; adding a first amount of the deprotection reagent to the second reactor; adding a second amount of the deprotection reagent to the first reactor; removing the first amount of the deprotection reagent from the second reactor; removing the second amount of the deprotection reagent from the first reactor; adding a second amount of the deprotection reagent to the second reactor; removing the second amount of the deprotection reagent from the second reactor; adding a first amount of an amino acid "X" activated ester to the first reactor; removing the first amount of the amino acid "X" activated ester from the first reactor; adding a first amount of the amino acid "X" activated ester to the second reactor; adding a second amount of the amino acid "X" activated ester to the first reactor; removing the first amount of the amino acid "X" activated ester from the second reactor; removing the second amount of the amino acid "X" activated ester from the first reactor; adding a second amount of the amino acid "X" activated ester to the second reactor; and removing the second amount of the amino acid "X" activated ester from the second reactor.

18. The method of claim 17, wherein the amino acid "X" present in the first and second amounts of the amino acid "X" activated ester itself has a protected N-group.

19. The method of claim 16 or 17, further comprising: adding a first amount of a deprotection reagent to a third reactor, wherein this addition is made after removing the first amount of the deprotection reagent from the second reactor, wherein the third reactor contains an amount of a protected N-group attached to a peptide synthesis resin; removing the first amount of the deprotection reagent from the third reactor; adding a second amount of the deprotection reagent to the third reactor, wherein this addition is made after removing the second amount of the deprotection reagent from the second reactor; adding a third amount of the deprotection reagent to the first reactor, wherein this third amount of the deprotection reagent is added to the first reactor after the second amount of the deprotection reagent has been removed from the first reactor; and transferring the third amount of the deprotection reagent from the first reactor to the second reactor, wherein this transfer is made after removing the second amount of the deprotection reagent from the second reactor; transferring the third amount of the deprotection reagent from the second reactor to the third reactor. ​ ​ adding a first amount of an amino acid "X" activated ester to the third reactor, wherein the adding is performed after removing the first amount of the amino acid "X" activated ester from the second reactor; removing the first amount of the amino acid "X" activated ester from the third reactor; adding a second amount of the amino acid "X" activated ester to the third reactor, wherein the adding is performed after removing the first amount of the amino acid "X" activated ester from the third reactor; and removing the second amount of the amino acid "X" activated ester from the third reactor.

20. The method of claim 19, further comprising: adding a third amount of the amino acid "X" activated ester to the first reactor, wherein the adding is performed after removing the second amount of the amino acid "X" activated ester from the first reactor; transferring the third amount of the amino acid "X" activated ester to the second reactor, wherein the transferring is performed after removing the second amount of the amino acid "X" activated ester from the second reactor; transferring the third amount of the amino acid "X" activated ester from the second reactor to the third reactor; removing the third amount of the amino acid "X" activated ester from the third reactor; adding the first amount of the amino acid "X" activated ester back to the first reactor, wherein the adding of the first amount of the amino acid "X" activated ester to the first reactor is performed after removing the first amount of the amino acid "X" activated ester from the third reactor.

21. A method of coupling an amino acid "X" to a deprotected N- group attached to a peptide synthesis resin, comprising: obtaining a first reactor and a second reactor, each of the first reactor and the second reactor containing an amount of a deprotected N- group attached to a peptide synthesis resin; adding a first amount of an amino acid "X" activated ester to the first reactor; removing the first amount of the amino acid "X" activated ester from the first reactor; adding the first amount of the amino acid "X" activated ester to the second reactor; adding a second amount of the amino acid "X" activated ester to the first reactor; removing the first amount of the amino acid "X" activated ester from the second reactor; removing the second amount of the amino acid "X" activated ester from the first reactor; adding the second amount of the amino acid "X" activated ester to the second reactor; and removing the second amount of the amino acid "X" activated ester from the second reactor.

22. The method of claim 21, further comprising washing the first reactor and the second reactor with a solvent.

23. The method of claim 20, further comprising adding a first amount of an amino acid "X" activated ester to a third reactor, wherein the adding is performed after removing the first amount of the amino acid "X" from the second reactor, wherein the third reactor contains an amount of a protected N- group attached to a peptide synthesis resin; removing the first amount of the amino acid "X" activated ester from the third reactor; and adding a second amount of the amino acid "X" to the third reactor.

24. The method of claim 23, further comprising: adding a third amount of the amino acid "X" to the first reactor, wherein the third amount of the amino acid "X" is added to the first reactor after the second amount of the amino acid "X" has been removed from the first reactor; and transferring a third amount of the amino acid "X" from the first reactor to the second reactor, wherein such transfer occurs after removal of the second amount of the amino acid "X" from the second reactor.

25. The method of claim 24, further comprising: removing a second amount of the deprotection reagent from the first reactor; transferring a third amount of the deprotection reagent from the first reactor to the second reactor, wherein such transfer occurs after removal of the second amount of the deprotection reagent from the second reactor.

30. The method of claim 29, further comprising: removing a second amount of the deprotection reagent from the third reactor; transferring a third amount of the deprotection reagent from the second reactor to the third reactor; removing a third amount of the deprotection reagent from the third reactor; and adding the first amount of the deprotection reagent back to the first reactor, wherein the first amount of the deprotection reagent is added back to the first reactor after the first amount of the deprotection reagent has been removed from the third reactor.

26. A method of deprotecting a protected N-group attached to a peptide synthesis resin, comprising: obtaining a first reactor and a second reactor, each of the first reactor and the second reactor containing an amount of a protected N-group attached to a peptide synthesis resin; adding a first amount of a deprotection reagent to the first reactor; removing the first amount of the deprotection reagent from the first reactor; adding the first amount of the deprotection reagent to the second reactor; adding a second amount of the deprotection reagent to the first reactor; removing the first amount of the deprotection reagent from the second reactor; removing the second amount of the deprotection reagent from the first reactor; adding the second amount of the deprotection reagent to the second reactor; and removing the second amount of the deprotection reagent from the second reactor.

27. The method of claim 26, further comprising washing the first reactor and the second reactor with a solvent.

28. The method of claim 26, further comprising: adding a first amount of the deprotection reagent to a third reactor, wherein such addition occurs after removal of the first amount of the deprotection reagent from the second reactor, wherein the third reactor contains an amount of a protected N-group attached to a peptide synthesis resin; removing the first amount of the deprotection reagent from the third reactor; and adding a second amount of the deprotection reagent to the third reactor, wherein such addition occurs after removal of the second amount of the deprotection reagent from the second reactor.

29. The method of claim 28, further comprising: adding a third amount of the deprotection reagent to the first reactor, wherein such third amount of the deprotection reagent is added to the first reactor after the second amount of the deprotection reagent has been removed from the first reactor; and transferring the third amount of the deprotection reagent from the first reactor to the second reactor, wherein such transfer occurs after removal of the second amount of the deprotection reagent from the second reactor.

30. The method of claim 29, further comprising: removing a second amount of the deprotection reagent from the third reactor; transferring a third amount of the deprotection reagent from the second reactor to the third reactor; removing a third amount of the deprotection reagent from the third reactor; and adding the first amount of the deprotection reagent back to the first reactor, wherein the first amount of the deprotection reagent is added back to the first reactor after the first amount of the deprotection reagent has been removed from the third reactor.

31. The method of claim 9, wherein a plurality of wash cycles occurs between the addition of each particular amino acid.

32. The method of claim 31, wherein the wash solvent from each wash cycle is collected in a recirculation vessel and the recirculated wash solvent is used in the next wash cycle.

33. The method of claim 32, wherein the recirculated wash solvent is used for the first half of the subsequent wash cycle.

34. The method of claim 32, wherein the use of the recirculated wash solvent reduces solvent requirements by one-half.

35. The method of claim 32, wherein the use of the recirculated wash solvent reduces solvent requirements by about 79%.

36. The method of claim 9, wherein the solvent comprises an environmentally friendly or green solvent.

37. The method of claim 36, wherein the environmentally friendly or green solvent is acetonitrile, ethyl acetate, isopropyl acetate, 2-methyltetrahydrofuran, cyclopentyl methyl ether, or N-butyl pyrrolidone, or a mixture thereof.

38. The method of claim 9, wherein the solvent is a mixture of N-butyl pyrrolidone and tetrahydrofuran.

39. The method of claim 9, wherein the solvent is a mixture of N-butyl pyrrolidone and 2-methyltetrahydrofuran.

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