Solid Phase Peptide Synthesis (SPPS) Processes and Related Systems

Abstract

A process for deprotecting amino acids during solid phase peptide synthesis includes removing a protecting group of a protected amino acid with a deprotecting agent in a reaction vessel, during which at least a portion of the deprotecting agent is volatilized or evaporated into an upper interior of the reaction vessel. The process further includes directing an inert gas through the interior of the reaction vessel to purge the volatilized deprotecting agent from the interior of the reaction vessel. The present disclosure also relates to a solid phase peptide synthesis system for deprotecting amino acids.

Description

[Technical field]

[0001] CROSS REFERENCE TO PRIORITY APPLICATION This application claims priority to U.S. Provisional Patent Application No. 63 / 245,674, filed September 17, 2021, the disclosure of which is incorporated by reference in its entirety.

[0002] This application also claims priority to U.S. Provisional Patent Application No. 63 / 401,349, filed August 26, 2022, the disclosure of which is incorporated by reference in its entirety.

[0003] Field The present disclosure relates to solid phase peptide synthesis processes and related systems. [Background technology]

[0004] background Since its inception in 1963, solid phase peptide synthesis has been the primary means available for peptide synthesis. J. Am. Chem. Soc. 1963, 85, 14, 2149-2154. Solid phase peptide synthesis (also called "SPPS") is a process used to chemically synthesize peptides on a solid support, such as a solid-phase resin. SPPS involves a repeated series of steps to combine amino acids to form a peptide.

[0005] In the SPPS process, the first amino acid is usually attached to the solid support by a linking group at its carboxyl or C-terminus. The first amino acid also usually contains a protecting group at its amine or N-terminus to protect against undesired reactions.

[0006] The deprotecting agent removes the protecting group (the first amino acid is "deprotected"), so that a second amino acid can be coupled via its acid group to the amine group of the first acid. The second (and subsequent) acids are also initially protected.

[0007] Thus, the general flow of solid phase peptide synthesis is to deprotect, couple, and repeat until the desired peptide is complete, which is then cleaved from the solid phase resin.

[0008] SPPS dramatically simplified solution-based peptide synthesis, providing a framework for building peptide chains one amino acid at a time through repeated deprotection and coupling steps, with easy isolation by simple filtration as opposed to more tedious extraction processes after each deprotection and coupling step.

[0009] Traditionally, SPPS processes require multiple washing steps between the deprotection and coupling steps to remove residual deprotection agent and minimize peptide impurities. Residual deprotection agent may, for example, prematurely remove the protecting group (e.g., Fmoc protecting group) from the amino acid that is to be coupled to the deprotected amino acid. This may result in the undesired insertion of additional amino acids into the growing peptide chain. Residual deprotection agent may also reduce the activity of the amino acid by reacting with it, which may result in the deletion of the peptide chain. This may result in both the insertion and deletion of the next amino acid, which may result in impurities that are extremely difficult to separate. For this reason, extensive washing (e.g., multiple washing steps) is typically employed after any deprotection step to prevent these potentially difficult-to-separate impurities.

[0010] Multiple washing steps can improve the purity of shorter peptides, but even with multiple washing steps, the formation of impurities remains a problem, especially as the number of amino acids in the peptide chain increases.

[0011] Furthermore, compared to solution phase synthesis, SPPS can result in significant waste generation from successive washing steps between each deprotection and coupling step. Historically, this involved approximately five washes between each step, resulting in approximately 80-90% of the total waste being generated from the washes. Chan, WC, & White, PD, Fmoc solid phase peptide synthesis: A practical approach. New York: Oxford University Press (2000). Thus, there is a need for solid phase peptide synthesis processes and systems that can provide peptide sequence purity, including improved purity for longer peptide sequences having, for example, up to 50, 100 or more amino acid derived units, improve process efficiency, and / or minimize or eliminate post-deprotection washes, thereby reducing the amount of solvent required for the SPPS process and associated material costs, solvent disposal issues, etc. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] J. Am. Chem. Soc. 1963, 85, 14, 2149-2154 [Non-Patent Document 2] Chan, WC, & White, PD, Fmoc solid phase peptide synthesis: A practical approach. New York: Oxford University Press (2000) Summary of the Invention [Means for solving the problem]

[0013] overview The present disclosure relates to a process for deprotecting a protected amino acid (e.g., as a step in solid phase peptide synthesis). Deprotecting a protected amino acid (deprotection reaction) removes the protecting group of the protected amino acid (deprotecting the amino acid) and prepares the amino acid for a coupling reaction with a second amino acid.

[0014] Generally, the deprotection processes of the present disclosure use an inert gas to remove (vent, purge, release, transfer, replace, etc.) the volatilized deprotection agent from the reaction vessel. In various embodiments, the deprotection processes of the present disclosure include the step of directing an inert gas through a portion of the interior of the reaction vessel containing the volatilized deprotection agent (e.g., directing an inert gas through a headspace of the reaction vessel containing the volatilized deprotection agent) to remove (vent, purge, release, transfer, replace, etc.) the volatilized deprotection agent from the interior of the reaction vessel.

[0015] In a first embodiment, a process for deprotecting a protected amino acid (e.g., during solid-phase peptide synthesis) includes heating the protected amino acid and the deprotecting agent in a reaction vessel (e.g., in the lower interior of the reaction vessel) during the deprotection reaction. The deprotecting agent may generally have a lower boiling point compared to the temperature used to heat the protected amino acid and the deprotecting agent and / or compared to the boiling point of the solvent that may be present, such as dimethylformamide (DMF) and N-methylpyrrolidinone (NMP). Thus, the deprotecting agent volatilizes or vaporizes during the heating step of the deprotection process (e.g., the deprotecting agent volatilizes or vaporizes in the upper interior of the reaction vessel).

[0016] In a first embodiment, the process for deprotecting a protected amino acid further comprises the step of directing (e.g., continuously and / or intermittently) an inert gas through the interior of the reaction vessel during the heating step to remove (purge, vent, release, displace, replace, etc.) the volatilized deprotection agent from the interior of the reaction vessel. More specifically, in a first embodiment, the process may comprise the steps of directing (introducing, feeding, etc.) an inert gas into the upper interior (e.g., headspace) of the reaction vessel containing the volatilized deprotection agent through a first opening located at the top of the reaction vessel, and venting (venting) the inert gas and the evaporated deprotection agent from the upper interior (e.g., from the headspace) of the reaction vessel through a second opening also located at the top of the reaction vessel. Thus, in a first embodiment, the process may include the steps of removing (purging, venting, releasing, displacing, removing, etc.) the volatilized deprotection agent from the upper interior of the reaction vessel (e.g., from the headspace of the reaction vessel) by directing an inert gas into the upper interior of the reaction vessel through a first opening located at the top of the reaction vessel, through the upper interior of the reaction vessel containing the vaporized deprotection agent (e.g., through the headspace), and out of the upper interior of the reaction vessel (e.g., out of the headspace) through a second opening also located at the top of the reaction vessel.

[0017] In a second embodiment, a process for deprotecting a protected amino acid (e.g., during solid phase peptide synthesis) includes removing a protecting group of the protected amino acid with a deprotection composition, the deprotection composition comprising a deprotecting agent in an amount of about 5 vol% or less based on the total volume of the deprotection composition. For example, the deprotection composition may comprise a deprotecting agent in an amount of about 2 vol% to about 5 vol%, such as about 2 to about 4.5 vol%, such as about 3 to about 4.5 vol%, as another example, about 3.5 to about 4.5 vol%, based on the total volume of the deprotection composition.

[0018] In a second embodiment, the protected amino acid and the deprotection composition are present in the lower interior of the reaction vessel. The deprotection agent generally has a lower boiling point compared to the deprotection reaction temperature and / or the temperature of the reaction vessel during deprotection, and / or a lower boiling point compared to the boiling point of the solvent(s) that may be present in the reaction vessel (e.g., the solvents present in the deprotection composition), such as dimethylformamide (DMF) and N-methylpyrrolidinone (NMP). Thus, at least a portion of the deprotection agent evaporates (volatilizes) into the upper interior of the reaction vessel during the removal step.

[0019] The process of the second embodiment may also include, during the step of removing the protecting group, a step of directing (e.g., continuously and / or intermittently) an inert gas through the interior of the reaction vessel to remove (bleed, release, purge, displace, replace, etc.) evaporated (volatilized) deprotecting agent from the upper interior of the reaction vessel.

[0020] In a second embodiment, the directing step may include directing (introducing, feeding, etc.) an inert gas into an upper interior (e.g., headspace) of the reaction vessel containing the vaporized deprotection agent, and discharging (venting) the inert gas and vaporized deprotection agent from the upper interior (e.g., headspace) of the reaction vessel. More specifically, the directing step may include directing the inert gas through a first opening located at the top of the reaction vessel and through the upper interior (e.g., headspace) of the reaction vessel containing the vaporized deprotection agent to the upper interior (e.g., headspace) of the reaction vessel, and discharging (venting) the inert gas and vaporized deprotection agent from the upper interior (e.g., from the headspace) of the reaction vessel through a second opening also located at the top of the reaction vessel.

[0021] In the second embodiment, as another example, the directing step may include directing (introducing, feeding, etc.) an inert gas into the lower interior of the reaction vessel, and discharging (discharging) the inert gas and evaporated deprotection agent from the upper interior (e.g., head space) of the reaction vessel. More specifically, the directing step may include directing the inert gas into the lower interior of the reaction vessel through an opening located at the bottom of the reaction vessel, and upward from the lower interior of the reaction vessel (e.g., upward through the reactants in the lower interior of the reaction vessel) through the upper interior (e.g., head space) of the reaction vessel including the evaporated deprotection agent, and discharging (discharging) the inert gas and evaporated deprotection agent from the upper interior (e.g., head space) of the reaction vessel through another opening located at the top of the reaction vessel.

[0022] In the second embodiment, as yet another example, the directing step may include directing (introducing, supplying, etc.) an inert gas to both an upper interior (e.g., headspace) of the reaction vessel containing the vaporized deprotection agent and a lower interior of the reaction vessel, as well as releasing (discharging) the inert gas and the vaporized deprotection agent from the upper interior (e.g., headspace) of the reaction vessel. More specifically, the directing step may include directing the inert gas through a first opening located at the top of the reaction vessel and through an upper interior (e.g., headspace) of the reaction vessel containing evaporated deprotection agent, and directing the inert gas through a second opening located at the bottom of the reaction vessel to a lower interior of the reaction vessel and upward from the lower interior of the reaction vessel (e.g., upward through the reactants in the lower interior of the reaction vessel) through the upper interior (e.g., headspace) of the reaction vessel containing evaporated deprotection agent, and releasing (venting) the inert gas and evaporated deprotection agent from the upper interior (e.g., headspace) of the reaction vessel through a third opening located at the top of the reaction vessel.

[0023] A second embodiment may include heating the protected amino acid and the deprotection composition during the step of removing the protecting group from the protected amino acid, where at least a portion of the deprotection agent evaporates into an upper interior of the reaction vessel during the heating step, and directing an inert gas through the interior of the reaction vessel during the heating step to remove (vent, bleed, etc.) the inert gas and evaporated deprotection agent from the upper interior of the reaction vessel. Alternatively, in the second embodiment, the step of removing the protecting group may be performed at room temperature.

[0024] Except for the variations noted and variations that will be apparent to those skilled in the art, the various embodiments of the present disclosure may be similar. However, the present invention may be embodied in many different forms and should not be construed as being limited to the embodiments described herein. For example, features disclosed as part of one embodiment may be used in conjunction with another embodiment to obtain a further embodiment. Hereinafter, some of the embodiments are specified sequentially, but the order does not relate to relative priority.

[0025] If used, the heating step in any of the embodiments of the deprotection process disclosed herein may be performed at a temperature of, for example, about 40° C. to about 120° C., in another example about 50° C. to about 120° C., and in another example about 70° C. to about 120° C. The heating step in any of the embodiments of the deprotection process disclosed herein may be performed using microwave irradiation.

[0026] The present disclosure also relates to a process for solid phase peptide synthesis (SPPS), which includes deprotecting a first protected amino acid according to any of the embodiments described herein (e.g., the first embodiment and / or the second embodiment of the deprotection step) to provide a deprotected amino acid, and coupling a second amino acid to the deprotected amino acid to form a peptide from the first and second amino acids.

[0027] In some embodiments (e.g., the second embodiment), the SPPS process may not include a washing step after the deprotection step and before the coupling step. In some embodiments (e.g., the second embodiment), the SPPS process may include a washing step after the deprotection step and before the coupling step, using a washing composition (e.g., a solvent) in an amount less than the total volume of the deprotection composition. For example, the washing step may include washing using a washing composition in an amount less than 1 / 2 the total volume of the deprotection composition, and as another example, washing using a washing composition in an amount less than 1 / 3 the total volume of the deprotection composition.

[0028] The present disclosure also relates to a system for solid phase peptide synthesis.

[0029] The present disclosure can provide a variety of unexpected benefits. The disclosed process can provide better (e.g., improved) peptide purity for even longer peptides containing up to 50, 100, or more amino acids relative to peptide purity achieved using a SPPS process that does not use an inert gas to remove (bleed, purge, vent, etc.) the evaporated deprotection agent from the reaction vessel as described herein. The disclosed process also allows for the use of more reactive deprotection agents with lower boiling points (e.g., pyrrolidine) at higher temperatures, which can speed up reaction times while minimizing the deleterious effects associated with using low boiling, volatile reactants.

[0030] Thus, this process can improve peptide purity, even with long peptide chains, and despite significant evaporation observed when using pyrrolidine as a deprotecting agent due to its lower boiling point. This process can also provide the benefit of faster deprotection rates.

[0031] The disclosed processes can also provide effective SPPS results (e.g., as measured by peptide purity) using relatively small amounts of deprotecting agent (e.g., about 5 vol.% or less relative to the total volume of the deprotected composition), and / or without the need for a washing step after deprotection and before conjugation, and / or with a washing step after deprotection and before conjugation that uses reduced amounts of solvent (e.g., using an amount of solvent less than the total volume of the deprotected composition). This can provide, for example, improved process efficiency, energy savings, reduced amounts of solvent required for the SPPS process, reduced material costs, reduced solvent disposal issues, and other benefits.

[0032] The above illustrative summary, as well as other exemplary embodiments, objects, and / or advantages of the present invention, and the manner in which they are achieved, are further described in the following detailed description and the accompanying drawings. The above summary provides some brief examples and is not exhaustive. The present invention is not limited to the above examples.

[0033] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures. These figures are schematic and are not intended to be drawn to scale. In these figures, each identical or nearly identical component shown is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, and not every component of each embodiment of the present invention is shown unless explanation is necessary to allow a person skilled in the art to understand the present invention. The present invention may be embodied in many different forms and should not be construed as being limited to the examples shown in the figures. [Brief description of the drawings]

[0034] [Figure 1A] FIG. 1A is a cross-sectional view of an exemplary reaction vessel, illustrating generally a process for deprotecting a protected amino acid according to an embodiment of the present disclosure.

[0035] [Figure 1B]FIG. 1B is a cross-sectional view of another exemplary reaction vessel, illustrating generally a process for deprotecting a protected amino acid according to another embodiment of the present disclosure.

[0036] [Figure 2A] FIG. 2A is a schematic flow diagram illustrating selected portions of an exemplary peptide synthesis system according to an embodiment of the present disclosure.

[0037] [Figure 2B] FIG. 2B is a schematic flow diagram illustrating selected portions of an exemplary peptide synthesis system according to another embodiment of the present disclosure.

[0038] [Figure 3A] FIG. 3A is an ultra-performance liquid chromatography (also referred to herein as "UPLC") chromatograph of thymosin synthesized by solid-phase peptide synthesis utilizing headspace purging (e.g., continuous headspace cleaning) between the deprotection step(s) described in Example 1 according to an embodiment of the present disclosure.

[0039] [Figure 3B] FIG. 3B is a UPLC chromatograph of thymosin synthesized using solid-phase peptide synthesis without headspace purging (e.g., without continuous headspace cleaning) between the deprotection step(s) described in Comparative Example 1.

[0040] [Figure 4A] FIG. 4A is a UPLC chromatograph of proinsulin synthesized using solid phase peptide synthesis with headspace purging (e.g., continuous headspace cleaning) between deprotection step(s) as described in Example 2 according to an embodiment of the present disclosure.

[0041] [Figure 4B]FIG. 4B is a mass spectrum of proinsulin synthesized using solid phase peptide synthesis with headspace purging (e.g., continuous headspace cleaning) between deprotection step(s) as described in Example 2 according to an embodiment of the present disclosure.

[0042] [Figure 5A] FIG. 5A is a UPLC chromatograph of HIV-1 protease synthesized using solid phase peptide synthesis with headspace purging (e.g., continuous headspace cleaning) between deprotection step(s) as described in Example 3 according to an embodiment of the present disclosure.

[0043] [Figure 5B] FIG. 5B is a mass spectrum of HIV-1 protease synthesized using solid phase peptide synthesis with headspace purging (e.g., continuous headspace cleaning) between deprotection step(s) as described in Example 3 according to an embodiment of the present disclosure.

[0044] [Figure 6A] FIG. 6A is a chromatograph of a Barstar UPLC synthesized using solid phase peptide synthesis with headspace purging (e.g., continuous headspace cleaning) between the deprotection step(s) described in Example 4 according to an embodiment of the present disclosure.

[0045] [Figure 6B] FIG. 6B is a mass spectrum of Barstar synthesized using solid phase peptide synthesis with headspace purging (e.g., continuous headspace cleaning) between deprotection step(s) as described in Example 4 according to an embodiment of the present disclosure.

[0046] [Figure 7A]FIG. 7A is a UPLC chromatograph of MDM2 synthesized using solid phase peptide synthesis with headspace purging (e.g., continuous headspace cleaning) between deprotection step(s) as described in Example 5 according to an embodiment of the present disclosure.

[0047] [Figure 7B] FIG. 7B is a mass spectrum of MDM2 synthesized using solid phase peptide synthesis with headspace purging (e.g., continuous headspace cleaning) between deprotection step(s) as described in Example 5 according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Detailed Description The above and other aspects, features, and advantages of the present disclosure will become apparent from the following detailed description of the embodiments. It should be understood that the present invention is not limited to the following embodiments, and may be embodied in various ways by those skilled in the art without departing from the scope of the present invention. Rather, the embodiments are provided for complete disclosure and to provide a complete understanding of the present invention to those skilled in the art. The scope of the present invention should be defined only by the appended claims.

[0049] Embodiments of the present disclosure relate to processes and systems for deprotecting protected amino acids (e.g., as a step in solid phase peptide synthesis). In exemplary embodiments, the processes and systems are batch-based processes and systems.

[0050] Figure 1A is a schematic cross-sectional view of a reaction vessel suitable for use in the amino acid deprotection and peptide synthesis processes and systems according to embodiments of the present disclosure, and also shows a schematic of a process according to embodiments of the present disclosure for deprotecting a protected amino acid.

[0051] Figure IB is a schematic cross-sectional view of another reaction vessel suitable for use in the amino acid deprotection and peptide synthesis processes and systems according to embodiments of the present disclosure, and also shows a schematic of a process according to embodiments of the present disclosure for deprotecting a protected amino acid.

[0052] Unless otherwise indicated, elements shown in FIG. 1A have the same reference numbers as in other figures, including FIG. 1B.

[0053] 1A and 1B, reaction vessel 4 includes at least one sidewall 6 that extends around an interior 7 (e.g., cavity) of reaction vessel 4 (reaction vessel interior 7 is also referred to herein as an exterior interior space). The particular size and shape of reaction vessel 4 is not limited. Reaction vessels suitable for use in solid phase peptide synthesis are well known in the art and are commercially available.

[0054] The size (internal volume) of the reaction vessel is not limited. Exemplary reaction vessel sizes can range from less than 1 liter to 40 liters or more, such as 10 ml, 30 ml, 125 ml, 1 liter, 3 liters, 5 liters, 8 liters, 10 liters, 15 liters, etc., up to 40 liters or more, and are not limited thereto.

[0055] The reaction vessel 4 further comprises one or more openings. As a non-limiting example, Figure 1A shows openings 10, 12, and 14 located at the top of the reaction vessel 4 (e.g., in the top wall), as well as opening 16 located at the bottom of the reaction vessel 4 (e.g., in the bottom wall). As another non-limiting example, Figure 1B shows openings 200, 202, 204, 206, and 208 located at the top of the reaction vessel 4 (e.g., in the top wall), as well as opening 16 located at the bottom of the reaction vessel 4 (e.g., in the bottom wall). The openings (e.g., inlets, outlets, ports, etc.) allow for the introduction and / or removal of fluids (e.g., gases and / or liquids) and / or solids, such as reactants, solvents, gases, products (peptides), by-products, and excess (residual) reactants, as discussed in more detail herein.

[0056] Those skilled in the art will understand that the reaction vessel 4 is not limited to the number and / or locations of the openings shown in Figures 1A and 1B, and that other reaction vessel designs and configurations having fewer or more openings and / or different locations thereof may be used (e.g., the reaction vessel may include fewer or more openings located in the top wall and / or bottom wall and / or side wall, etc.).

[0057] As shown in FIG. 1A , fluids and / or solids can be introduced (e.g., moved, transported, directed, discharged, purged, drained, expelled, etc.) into and out of reaction vessel 4 via one or more flow paths (e.g., lines, passages, tubes, manifolds, etc.), such as flow paths 20, 22, and 24 in fluid communication with openings 10, 12, and 14, respectively, and flow path 26 in fluid communication with opening 16.

[0058] Other non-limiting examples of flow paths are shown in FIG. 1B as flow paths 210, 212, 214, 216, and 218 in fluid communication with openings 200, 202, 204, 206, and 208, respectively, and flow path 26 in fluid communication with opening 16.

[0059] In some embodiments, reaction vessel 4 may include at least one spray head (e.g., spray nozzle) or equivalent structure located inside the reaction vessel for adding (e.g., directing, delivering, spraying, etc.) fluids (e.g., solvents, reactants, and / or inert gases) into the reaction vessel. The spray head may be a component (e.g., a component, element, etc.) separate from the reaction vessel (e.g., capable of being attached to or detached from the reaction vessel) or may be an integral part of the reaction vessel.

[0060] As a non-limiting example, FIG. 1B illustrates an embodiment including a spray head 220 positioned within the interior space 7 (also referred to herein as the outer interior space) of the reaction vessel 4. The spray head 220 is positioned within the interior space 7 (also referred to herein as the outer interior space) of the reaction vessel 4 and includes at least one sidewall 220a extending around the periphery of the inner interior space. The spray head 220 includes a first portion (end) 220b proximal to the opening 208 and in fluid communication with the opening 208 (and the flow path 218), and a second portion (end) 220c distal to the opening 208. A plurality of holes 222 (e.g., ports, etc.) may extend at least partially around the periphery of the inner interior space and may be defined, for example, in the sidewall 220a. The inner interior space and the outer interior space are in fluid communication with each other by a plurality of holes.

[0061] The spray head 220 is configured such that a fluid (e.g., an inert gas, a solvent, etc., as discussed herein) directed into the inner interior space of the spray head 220 via the flow passage 218 and the opening 208 flows (e.g., is sprayed, directed, dispensed, etc.) through holes (e.g., one, more than one, a number, or all holes) of the plurality of holes 222 into the outer interior space 7 of the reaction vessel 4. In some embodiments, the spray head 220 can be configured such that the fluid exiting at least one or more holes of the plurality of holes 222 is directed (sprayed) toward the sidewall 6 of the reaction vessel. An exemplary spray pattern is shown in FIG. 1B, generally (e.g., approximately) by the dashed line 224, where the fluid exiting the holes of the plurality of holes 222 is directed at an approximately downward angle toward the sidewall 6.

[0062] The present disclosure is not limited with respect to the particular spray head configuration, location within the reaction vessel, spray pattern, and / or direction (angle) of fluid exiting the spray head bores as shown in Figure 1B, and other spray head configurations, locations, spray patterns, and / or spray angles, etc. Spray heads suitable for use in SPPS processes and systems are known in the art and can be used in the present disclosure.

[0063] The present disclosure is not limited to a particular number and / or location of openings and flow paths, and thus a reaction vessel may have one, two, three, four, or more openings and associated flow paths as desired. Additionally, any series of flow paths and associated valves that act to direct, allow, and / or block (e.g., close, restrict, etc.) the flow of fluids and / or solids may be used.

[0064] In the deprotection process of the present disclosure, reactants designated as 30 are present in the lower portion of reaction vessel 4. For example, the reactants may rest on a filter 32 located in the lower portion of reaction vessel 4. Filter 32 may also prevent a solid support (e.g., a solid resin support) as discussed herein from entering flow path 26.

[0065] Reactant 30 includes protected amino acids, ie, amino acids that include at least one protecting group attached to a functional group, such as the terminal amine group, to protect the functional group from undesired reactions.

[0066] Reactants 30 also include a deprotecting agent, which reacts with a protected amino acid to remove the protecting group and make the previously protected functional group (e.g., a terminal amine group) available to react (e.g., to form a peptide chain with one, two, or more consecutive amino acids).

[0067] The deprotecting agents used in the SPPS process are typically liquids at room temperature, and therefore are typically added and / or present in the reaction vessel as part of a deprotecting composition that includes the deprotecting agent (wherein the amount of the deprotecting agent is greater than zero volume percent based on the total volume of the deprotecting composition) and a suitable solvent.

[0068] The deprotecting agent may be an organic base. Examples of organic bases suitable for use as a deprotecting agent include, without limitation, piperidine and / or pyrrolidine. Other organic bases that provide the deprotecting function without otherwise interfering with other steps in the process, the growing peptide chain, or the system may also be suitable.

[0069] Examples of solvents that may be part of the deprotection composition include, without limitation, dimethylformamide (DMF), dimethylacetamide (DMA), N-methylpyrrolidinone (NMP), green solvents, and / or non-reprotoxin solvents, and the like, and combinations and / or mixtures thereof. Examples of green and / or non-reprotoxin solvents may include, without limitation, N-formylmorpholine (NFM), N-butylpyrrolidinone (NBP), alkoxybenzene-based solvents (e.g., anisole, dimethoxybenzene-based solvents such as 1,3-dimethoxoybenzene, and the like), and others, and combinations and / or mixtures thereof.

[0070] In an embodiment (e.g., the second embodiment) of the present disclosure, the deprotection composition may include a deprotection agent in an amount of about 5 volume percent (vol%) or less based on the total volume (100% volume) of the deprotection composition (the deprotection agent is present, i.e., the amount of the deprotection agent is greater than zero). In some embodiments (e.g., the second embodiment), the deprotection composition may include a deprotection agent in an amount of about 1 vol% to about 5 vol%, for example about 2 vol% to about 5 vol%, for example about 2 to about 4.5 vol%, for example about 3 to about 4.5 vol%, as another example, about 3.5 to about 4.5 vol%, based on the total volume of the deprotection composition. In some embodiments (e.g., the second embodiment), the deprotection composition may include a deprotection agent in an amount of greater than zero to about 4.5 vol%, based on the total volume of the deprotection composition. In some embodiments (e.g., the second embodiment), the deprotection composition may include a deprotection agent in an amount of greater than zero to about 4 vol%, based on the total volume of the deprotection composition. In some embodiments (e.g., the second embodiment), the deprotection composition may include a deprotection agent in an amount of from greater than zero to about 3 vol%, based on the total volume of the deprotection composition. The amount of deprotection agent (e.g., in the second embodiment) may be any value within the ranges described herein, including the endpoints (e.g., any value within the range of from greater than zero to about 5 vol%), and all subranges within the ranges are also disclosed.

[0071] However, the concentration of the deprotecting agent in the deprotecting composition is not so limited and may vary. For example, in some embodiments (e.g., the first embodiment) of the present disclosure, the deprotecting composition may include a deprotecting agent in an amount greater than about 5 vol%, such as about 20 vol% or more, based on the total volume (100% volume) of the deprotecting composition. In some embodiments (e.g., the first embodiment), the deprotecting composition may include a deprotecting agent in an amount of about 20 vol% to 100 vol%, such as about 20 to about 50 vol%, such as about 20 vol% to about 40 vol%, and as another example, about 20 to about 35 vol%, based on the total volume of the deprotecting composition. The amount of deprotecting agent (e.g., in the first embodiment) may be any value within the ranges described herein, including the endpoints (e.g., any value within the range of about 5 vol% to 100 vol%), and all subranges within the ranges are also disclosed.

[0072] Those skilled in the art will understand the meaning of the term amino acid. As used herein, the term amino acid in its broadest sense refers to organic compounds that contain both amine and carboxylic acid functional groups and in some cases also side chains. Those skilled in the art will also understand that amino acids include natural amino acids (proteinogenic amino acids) and / or non-proteinogenic amino acids, and will also understand the one-letter codes used to identify them.

[0073] The term peptide will also be understood by those of skill in the art. As used herein, the term peptide has its normal meaning in the art and can refer to an amide derived from two or more amino acids (identical or different) by bonding the carbonyl carbon of one amino acid to the nitrogen atom of another amino acid.

[0074] Suitable protective groups for use in the process of the present disclosure are well known in the art. Examples of suitable protective groups for protecting amine or N-terminus include, without limitation, fluorenylmethyloxycarbonyl (Fmoc) protective group.See, for example, Chan and White, Fmoc solid phase peptide synthesis, a practical approach, Oxford University Press (2000).

[0075] The amino acid may include a side chain protecting group. Examples of side chain protecting groups may include, without limitation, trityl, t-butyl, and / or 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) protecting groups. When the desired peptide chain length is obtained, the side chain protecting group can be removed.

[0076] The protected amino acid can be directly or indirectly bound to the solid support as known in the art. For example, the carboxy terminus of the protected amino acid can be bound to the solid support via a suitable linker. As another example, the carboxy terminus of the protected amino acid can be indirectly bound to the solid support, for example, by being bound to another amino acid (or to a peptide chain) which is then bound to the solid support.

[0077] Solid supports known in the art may be used in the process of the present disclosure.Non-limiting examples of solid support materials include polystyrene (e.g., in resin form such as microporous polystyrene resin, mesoporous polystyrene resin, macroporous polystyrene resin, etc.), glass, polysaccharides (e.g., cellulose, agarose), polyacrylamide resin, polyethylene glycol, and / or copolymer resin (e.g., including polyethylene glycol, polystyrene, etc.).In an exemplary embodiment, the solid support is a solid phase resin.

[0078] The solid support may have any suitable form, for example, the solid support may be in the form of beads, particles, fibers, and / or any other suitable form.

[0079] Those of skill in the art will understand how to attach an amino acid to a solid support (e.g., a solid-phase resin), and therefore, a detailed discussion of methods known in the art for attaching an amino acid (and / or a peptide containing two or more amino acids) to a solid support (e.g., a solid-phase resin) is not provided.

[0080] The deprotecting agent and the protected amino acid (directly or indirectly bound to a suitable solid support, such as a solid phase resin as discussed herein) can be mixed (combined) with a suitable solvent(s) as known in the art and can be in the form of a suspension (slurry) in a reaction vessel.

[0081] As noted herein, a deprotection reaction removes a protecting group from a protected group (e.g., a protected functional group) of a protected amino acid (also commonly referred to as the amino acid being deprotected).

[0082] In some embodiments (e.g., the second embodiment), the deprotection step may be performed without heating (e.g., at room temperature) so long as the deprotection conditions (type of base, time, etc.) are selected to facilitate evaporation of the deprotection agent into the headspace of the reaction vessel.

[0083] More typically, in some embodiments (e.g., the first and / or second embodiments), the deprotection process may include a step of heating the protected amino acid and / or the deprotection agent (e.g., heating the protected amino acid and / or the deprotection agent prior to delivery into reaction vessel 4 and / or heating the protected amino acid and / or the deprotection agent in reaction vessel 4 before and / or during the deprotection reaction). As used herein, reference to a deprotection agent may include the deprotection agent itself and / or a deprotection composition comprising the deprotection agent. That is, as used herein, reference to heating a deprotection agent may include heating the deprotection agent itself and / or heating a deprotection composition comprising the deprotection agent.

[0084] Heating during solid phase peptide synthesis can be useful, for example, to accelerate the rate of deprotection and thereby shorten the time required for peptide synthesis.

[0085] The heating temperature (e.g., the heating temperature of the protected amino acid and / or deprotection agent in the reaction vessel prior to delivery into the reaction vessel and / or prior to and / or during deprotection) can vary. In some embodiments, the heating step can be performed at a temperature of about 40° C. to about 120° C., for example, about 50° C. to about 120° C., for example, about 70° C. to about 120° C., for example, about 80° C. to about 120° C., for example, about 80° C. to about 110° C., for example, about 90° C. to about 110° C. In certain embodiments, the heating step can be performed at a temperature of about 70° C. to about 120° C., for example, about 90° C. to about 120° C., for example, about 90° C. to about 110° C. The temperature can be any value within the ranges described herein, including the endpoints (e.g., any value within the range of about 40° C. to about 120° C.), and all subranges within the ranges are also disclosed.

[0086] 1A and 1B show schematics of a heating step in which a heat source 40 heats the reaction vessel 4 and reactants 30. In certain embodiments, the heat source 40 includes a microwave source 42 positioned to direct microwave radiation 44 through a waveguide 46 attached to a microwave cavity (not shown) that contains the reaction vessel 4. The microwave power can be controlled as known in the art to provide a reaction temperature and / or reaction time (such as, without limitation, to provide a deprotection temperature as described herein and a deprotection reaction time in the range of about 10 seconds to about 15 minutes, as another non-limiting example, about 40 seconds to about 8 minutes).

[0087] In embodiments utilizing microwave energy to heat the reactants, reaction vessel 4 may be formed from a material that is transparent to microwave radiation, such as, but not limited to, glass, Teflon, and / or polypropylene.

[0088] Microwave sources are well known in the art and may include, for example, magnetrons, klystrons, and / or solid-state diodes. Suitable microwave sources, waveguides, and microwave cavities for solid-phase peptide synthesis processes and systems are well known in the art and are also commercially available (e.g., commercially available systems from CEM Corporation as discussed herein). Thus, one of ordinary skill in the art would know how to use them in solid-phase peptide synthesis processes and systems without undue experimentation.

[0089] However, the present disclosure is not limited to the use of a microwave source as the heat source, and other types of heat sources known in the art for solid phase peptide synthesis can be used.

[0090] Despite the benefits of heating, high temperatures during the deprotection step can present various challenges for peptide synthesis (including but not limited to the synthesis of longer peptides).

[0091] For example, the organic amine used in the deprotection reaction may have a relatively low boiling point compared to the boiling point of the solvent used in the deprotection reaction and / or the temperature of the deprotection step. Piperidine has a boiling point of about 106°C, and pyrrolidine has a boiling point of about 87°C. In contrast, the solvent dimethylformamide (DMF) has a boiling point of about 153°C, and the solvent N-methylpyrrolidinone (NMP) has a boiling point of about 200°C. Also in contrast, as noted herein, the deprotection reaction can be carried out at elevated temperatures, for example up to about 120°C, for example from about 90°C to about 120°C, as another example from about 90°C to about 110°C, without limitation.

[0092] The reaction vessel may exhibit a continuum of temperatures during processing, with the upper portion being at a lower temperature than the lower portion. Because the deprotection agent may have a boiling point lower than that of other reagents, such as the solvent, and / or a boiling point lower than the reaction temperature, the deprotection agent may volatilize (evaporate) to the upper portion of the reaction vessel (e.g., headspace) and condense on the upper portion of the sidewall(s) and / or on the top wall of the reaction vessel. The rate / amount of volatilization (evaporation) may also be increased, for example, if air is bubbled through the reactants during deprotection to aid in mixing the reactants.

[0093] Volatilization of the deprotecting agent can be particularly problematic when pyrrolidine is used. Pyrrolidine may provide faster deprotection than piperidine, making it a desirable deprotecting agent. Being a five-membered ring (versus a six-membered piperidine ring), the carbon atom of pyrrolidine is bent further back from the nitrogen atom, making it easier to attack for deprotection. However, pyrrolidine has a lower boiling point than piperidine, so significant evaporation and subsequent condensation can occur during the deprotection process, which can limit its use, including, for example, in the synthesis of long peptides.

[0094] In the disclosed process, the heating step volatilizes (evaporates) the deprotecting agent (eg, pyrrolidine) from the bottom of reaction vessel 4 upward to the top of reaction vessel 4 (eg, into the headspace above reactants 30).

[0095] Residual deprotection agent remaining in the reaction vessel during subsequent solid phase peptide synthesis steps (e.g., coupling steps) (e.g., residual deprotection agent condensed on the top(s) of the sidewall and / or the top wall of the reaction vessel) can cause problems. Residual deprotection agent can, for example, prematurely remove the protecting group from an amino acid that should be coupled to an already deprotected amino acid. This can lead to undesired insertions in the peptide chain. Residual deprotection agent can reduce an activated amino acid by reacting with this amino acid, leading to deletions in the peptide chain. Thus, typically, SPPS processes have required multiple washing steps after deprotection and before coupling, as discussed herein.

[0096] Traditionally, multiple washing steps are used to help remove residual deprotecting agent to minimize or prevent its participation in subsequent solid-phase peptide synthesis steps (e.g., coupling steps). However, as the length of the peptide increases, multiple washing steps become less effective at preventing undesired reactions and reducing impurities, which can make it difficult to synthesize longer peptides with acceptable purity for downstream applications.

[0097] In contrast to conventional processes, the process of the present disclosure can facilitate the production of longer peptides with acceptable purity levels for downstream applications. However, the present disclosure is not limited to the production of longer peptides, and can generally facilitate the production of peptides with acceptable purity levels for downstream applications regardless of the length of the peptide. Also in contrast to conventional processes, in some embodiments (e.g., the second embodiment), the process of the present disclosure can help eliminate the washing step(s) between the deprotection step and the coupling step, and / or reduce the amount of solvent required for the washing step(s) between the deprotection step and the coupling step of the SPPS process, and / or facilitate the use of a smaller amount of deprotection agent (base) compared to conventional SPPS processes. Again without limitation, in some embodiments (e.g., the first and / or second embodiments of the deprotection step), it is currently believed that peptide purity, and / or elimination of a washing step, and / or reduction in the amount of solvent for the washing step, and / or reduction in the amount of deprotection base, can be facilitated by directing (e.g., continuously and / or intermittently) an inert gas through a portion of the interior of the reaction vessel containing the evaporated deprotection agent during the deprotection step to remove (e.g., vent, release, drain, transfer, replace, purge, etc.) the evaporated (volatilized) deprotection agent from the interior of the reaction vessel (e.g., from the headspace of the reaction vessel) (e.g., directing an inert gas through the upper interior of the reaction vessel containing the evaporated deprotection agent (through the headspace above the reactants)).

[0098] More specifically, in various embodiments (e.g., the first and / or second embodiments) described herein, the deprotection step may include directing (introducing, feeding, etc.) an inert gas into the interior of the reaction vessel through one or more openings in the reaction vessel. For example, the inert gas may be directed into the upper and / or lower interior of the reaction vessel through one or more openings (entry ports) located at the top and / or bottom of the reaction vessel, respectively. The directing step may further include directing (moving, etc.) the inert gas through the upper interior of the reaction vessel (e.g., through the headspace above the reactants) containing the evaporated deprotection agent (and in some embodiments directing the inert gas upward from the lower interior into / through the upper interior or headspace containing the evaporated deprotection agent), and removing (e.g., venting, releasing, evacuating, moving, replacing, purging, etc.) the evaporated (volatilized) deprotection agent from the upper interior of the reaction vessel (e.g., from the headspace above the reactants) through one or more other openings (exit ports) located at the top of the reaction vessel.

[0099] In some embodiments, the directing step may include directing the inert gas through an upper interior of the reaction vessel containing the vaporized deprotection agent (e.g., through the headspace above the reactants) via a first opening located at the top of the reaction vessel, to the upper interior of the reaction vessel containing the volatilized deprotection agent (e.g., the headspace above the reactants), and removing (e.g., venting, releasing, evacuating, transferring, replacing, purging, etc.) the inert gas and the volatilized deprotection agent from the upper interior (e.g., from the headspace above the reactants) through a second opening also located at the top of the reaction vessel. In some embodiments, the directing step may include directing an inert gas into a lower interior of the reaction vessel via a first opening located at the bottom of the reaction vessel and into / through an upper interior of the reaction vessel (e.g., through the headspace above the reactants) containing the evaporated deprotection agent from the lower interior of the reaction vessel upward (e.g., upward through the reactants in the lower interior of the reaction vessel) to remove (e.g., vent, release, evacuate, displace, purge, etc.) the evaporated (volatilized) deprotection agent from the upper interior (e.g., from the headspace above the reactants) through a second opening located at the top of the reaction vessel. In some embodiments, the directing step may include directing the inert gas into an upper interior of the reaction vessel containing the volatilized deprotection agent (e.g., the headspace above the reactants) via a first opening located at the top of the reaction vessel and through the upper interior of the reaction vessel containing the vaporized deprotection agent (e.g., through the headspace above the reactants), and directing the inert gas into a lower interior of the reaction vessel through a second opening located at the bottom of the reaction vessel, the inert gas optionally traveling upward from the lower interior of the reaction vessel to / through the headspace of the reaction vessel, and removing (e.g., venting, releasing, draining, transferring, replacing, purging, etc.) the vaporized (volatilized) deprotection agent from the upper interior (e.g., from the headspace above the reactants) through a third opening located at the top of the reaction vessel.

[0100] In some embodiments, the inert gas may be continuously directed through the reaction vessel as a continuous flow. In some embodiments, the inert gas may be directed through the reaction vessel as an intermittent (e.g., pulsed) flow.

[0101] In some embodiments, the inert gas directed into and / or moving through the reaction vessel (e.g., moving through the headspace of the reaction vessel containing the volatilized deprotection agent) may have a pressure of about 1 psi to about 25 psi. In some embodiments, the inert gas directed into and / or moving through the reaction vessel (e.g., moving through the headspace of the reaction vessel containing the volatilized deprotection agent) may have a pressure of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 psi. In some embodiments, the inert gas directed into and / or moving through the reaction vessel may have a pressure ranging from about any of the above pressure values ​​to about any other of the above pressure values. The pressure of the inert gas, including the inert gas, directed into and / or moving through the upper and / or lower interior of the reaction vessel can be any value within the ranges described herein, including the endpoints (e.g., any value within the range of about 1 to about 25 psi), and all subranges within the ranges are also disclosed. The inert gas can also be directed through the interior of the reaction vessel (e.g., through the upper interior or headspace) during the deprotection step at a flow rate based on the time rate during which the inert gas substantially replaces (moves) the gas volume of the headspace. More specifically, the flow rate of the inert gas can be an amount (volume) of inert gas that allows (provides) substantial replacement (movement) of the volume of gas in the headspace region of the reaction vessel by the inert gas within a selected time (time rate) (e.g., resulting in substantial replacement of the volume of volatilized deprotection agent in the headspace region of the reaction vessel by the inert gas). For example, the flow rate of the inert gas may be an amount (volume) of inert gas that results in (enables) substantial replacement (displacement) of a volume of gas in the headspace region of the reaction vessel (e.g., the volume of volatilized deprotection agent in the headspace region) about every 1 to 20 seconds, for example, about every 5 to 10 seconds.One of ordinary skill in the art will know, without undue experimentation, how to determine and calculate a suitable inert gas flow rate to replace (displace) the volume of headspace gas (volatilized deprotection agent) in the reaction vessel within a time frame (time rate).

[0102] Without wishing to be bound by any explanation or theory, it is presently believed that directing a source of inert gas into (through) the headspace above the reactants during deprotection may cause a high air exchange rate in the gas above the reactants (headspace gas containing volatilized deprotection agent), thereby displacing the volatilized deprotection agent from the reaction vessel. This may shorten the residence time of the volatilized deprotection agent in the reaction vessel, so that the volatilized deprotection agent is less likely to condense on the side and / or top walls of the vessel and can be removed more quickly. This may reduce the amount of residual deprotection agent remaining in the reaction vessel after the deprotection step is complete. The inert gas may also provide a downward force on droplets (e.g., condensed deprotection agent) on the side walls of the reaction vessel 4, thereby blowing the droplets toward the reactants 30 at the bottom of the reaction vessel 4.

[0103] Furthermore, in some embodiments (e.g., the second embodiment) in which the deprotection composition includes a small amount of deprotection agent (about 5 vol.% or less of the deprotection agent based on the total volume of the deprotection composition), the deprotection agent (e.g., pyrrolidine) may be essentially completely removed from the reaction vessel upon completion of the deprotection step. For example, without being bound by any theory or explanation, it is presently believed that in such embodiments, the deprotection agent may be substantially completely evaporated from the deprotection composition during the heating step, and / or the volatilized deprotection agent may be substantially completely removed from the headspace by venting an inert gas, each as described herein. Also without being bound by any theory or explanation, it is presently believed that in such embodiments, any residual amount of deprotection agent remaining after the deprotection step is completed, also as described in more detail herein, is sufficiently small to minimize issues associated with the presence of residual deprotection agent in the subsequent coupling step, even without a washing step after the deprotection step and / or even with a washing step using a reduced amount of washing solution (e.g., solvent) after the deprotection step.

[0104] In contrast to conventional approaches, the process of the present disclosure can facilitate the production of longer peptides with acceptable purity levels for downstream applications, since the amount of residual deprotection agent can be reduced. For example, the process can be useful for the production (e.g., batch SPPS synthesis) of longer peptides, including but not limited to peptides that contain 20 or more amino acid-derived units, such as 25 or more amino acid-derived units, in another example, 30 or more amino acid-derived units, in another example, 40 or more amino acid-derived units, in another example, 50 or more amino acid-derived units, in another example, 75 or more amino acid-derived units, in another example, 100 or more amino acid-derived units, in another example, 125 or more amino acid-derived units, and in another example, 150 or more amino acid-derived units. In some embodiments, the process is carried out to produce longer peptides, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 , 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 , 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or more amino acid derived units.However, the present disclosure is not limited to the production of peptides containing 20 or more amino acid derived units and may be used for the production (e.g., batch SPPS synthesis) of peptides containing fewer than 20 amino acid derived units (e.g., peptides containing 2 or more, e.g., 2-20, or more, amino acid derived units), which peptides may have an acceptable purity level for downstream applications.

[0105] The inert gas can be nitrogen. The present disclosure is not limited to the use of nitrogen as the inert gas, and other inert gases can be used, such as noble gases that have limited or no chemical interference with solid phase peptide synthesis reactions and systems.

[0106] In certain embodiments, as shown in FIG. 1A, the process may include providing pressurized inert gas from an inert gas source (e.g., an inert gas source designated 100 in FIG. 2A) through a flow path 20 and directing the pressurized inert gas through an opening 10 and through an upper interior of the reaction vessel 4 (e.g., through a headspace above the reactants 30) to an upper portion of the interior of the reaction vessel 4 containing the volatilized (evaporated) deprotection agent (e.g., directing the pressurized inert gas generally downward). As the pressurized inert gas flows through the upper interior of the reaction vessel 4 containing the volatilized (evaporated) deprotection agent (e.g., through a headspace above the reactants), the inert gas purges (e.g., vents, displaces, displaces, releases, vents, etc.) the volatilized deprotection agent from the interior of the reaction vessel 4 through opening 14 and into flow path 24. In this manner, the pressurized inert gas effectively displaces the volatilized deprotection gas from the headspace of the reaction vessel. This can reduce residence time and minimize condensation of the deprotection agent on the walls of the reaction vessel.

[0107] Gas flows (movements) within reaction vessel 4 are shown generally by arrows in FIG. 1A, including the upward flow of volatilized deprotecting agent (e.g., pyrrolidine) from reactants 30 in the lower interior of reaction vessel 4 to the headspace above the reactants (e.g., into the upper interior of reaction vessel 4), the downward flow of inert gas from the upper interior of reaction vessel 4 (e.g., through the headspace), and the purging (e.g., venting, transferring, releasing, etc.) of volatilized deprotecting agent and inert gas from the upper interior of reaction vessel 4 (e.g., from the headspace).

[0108] In certain embodiments, as shown in Figure IB, the process may include providing (e.g., directing) pressurized inert gas from an inert gas source (e.g., an inert gas source designated 100 in Figure 2B) through a flow path 218, through an opening 208, through an inner interior space of a spray head 220, and out an opening 222 to an outer interior space 7 of the reaction vessel 4 (e.g., to a headspace above the reactants 30). Figure IB also illustrates an embodiment in which the spray head 220 directs (e.g., sprays) the inert gas through the opening 222 toward a sidewall 6 of the reaction vessel 4 at an angle (e.g., a spray pattern) indicated generally by dashed line 224. This allows the inert gas to contact the sidewall and facilitate a cleaning effect that "washes" condensed deprotection agent toward the reactants at the bottom inside the reaction vessel 4.

[0109] As the pressurized inert gas flows through the upper interior of reaction vessel 4 (e.g., through the headspace above the reactants) containing the volatilized (evaporated) deprotection agent, the inert gas purges (e.g., vents, displaces, displaces, releases, vents, etc.) the volatilized deprotection agent from reaction vessel 4 through opening 206 and into flow path 216. Again, the pressurized inert gas effectively displaces the volatilized deprotection gas from the headspace of the reaction vessel, thereby reducing residence time and minimizing condensation of the deprotection agent on the walls of the reaction vessel.

[0110] Gas flows (movements) within reaction vessel 4 are shown generally by arrows and dashed lines in FIG. 1B, including the upward flow of volatilized deprotecting agent (e.g., pyrrolidine) from reactants 30 in the lower interior of reaction vessel 4 to the headspace above the reactants (e.g., into the upper interior of reaction vessel 4), the flow of inert gas from spray head 220 through the openings of multiple openings 222 (e.g., angled flow through the openings of multiple openings 222 into the headspace and through the headspace toward sidewall 6), and the purging (e.g., venting, transferring, releasing, etc.) of volatilized deprotecting agent and inert gas from the upper interior of reaction vessel 4 (e.g., from the headspace).

[0111] In certain embodiments, the process may include directing an inert gas to a lower portion (e.g., lower interior) of the reaction vessel 4 in addition to or as an alternative to directing the inert gas to an upper interior (e.g., headspace) of the reaction vessel through an opening in the top of the reaction vessel as described herein. For example, with reference to FIGS. 1A and 1B, the process may include directing pressurized inert gas from an inert gas source (which may be the same or different from the inert gas source of the inert gas introduced to the upper interior of the reaction vessel, if present) through a flow path 26 to introduce the pressurized inert gas into the lower interior of the reaction vessel 4 through opening 16. The inert gas may flow upward from the lower interior of the reaction vessel (e.g., upward through reactants 30) into / through the upper interior (e.g., headspace) of the reaction vessel 4 containing the volatilized (evaporated) deprotection agent. As the inert gas flows upward, it can purge (e.g., vent, displace, replace, release, vent, etc.) volatilized deprotection agent from the upper interior (e.g., headspace) of reaction vessel 4 through opening 14 and into flow path 24. Again, in this manner, the inert gas can displace volatilized deprotection gas from the headspace of the reaction vessel, thereby reducing residence time and minimizing condensation of the deprotection agent on the walls of the reaction vessel.

[0112] A flow of inert gas at the bottom of the reaction vessel can also agitate (mix, bubble, etc.) the reaction 30 of the protected amino acid and deprotecting agent.

[0113] In some embodiments, the process may include introducing (directing) both a first pressurized inert gas into the upper interior (e.g., headspace) of the reaction vessel containing the evaporated deprotection agent and a second pressurized inert gas into the lower interior of the reaction vessel. As a non-limiting example, with reference to Figures 1A and 1B, the process may include directing the first pressurized inert gas into the upper interior of the reaction vessel 4 through a first opening located at the top of the reaction vessel, e.g., opening 10 in Figure 1A or openings 208 and 222 in Figure 1B, and directing the second pressurized inert gas into the lower interior of the reaction vessel 4 through a second opening located at the bottom of the reaction vessel, e.g., opening 16 in Figures 1A and 1B. The first pressurized inert gas may travel (e.g., through the headspace) through the upper interior of the reaction vessel containing the evaporated deprotection agent, and the second pressurized inert gas may travel generally upward from the lower interior of the reaction vessel through reactant 30 into / through the upper interior (e.g., headspace) of the reaction vessel containing the evaporated deprotection agent. The first pressurized inert gas and optionally the second pressurized inert gas may purge (e.g., vent, transfer, displace, release, vent, etc.) volatilized deprotection agent from the upper interior (e.g., from the headspace) of the reaction vessel, for example through a third opening located at the top of the reaction vessel, for example opening 14 or 206 in Figures 1A and 1B, respectively.

[0114] A first inert gas (also referred to herein as an overhead inert gas) directed into and / or traveling through the upper interior (e.g., headspace above reactants 30) of reaction vessel 4 (e.g., a first inert gas directed through a first opening located at the top of the reaction vessel, e.g., opening 10 in FIG. 1A or openings 208 and 222 in FIG. 1B) may have a higher pressure than a second inert gas introduced into a lower portion of the interior of reaction vessel 4. As a non-limiting example, the first (overhead) inert gas directed into and / or traveling through the upper interior (e.g., traveling through the headspace) of reaction vessel 4 may have a pressure of from about 1 psi to about 25 psi. In some embodiments, the first (overhead) inert gas directed to and / or traveling through the upper interior of the reaction vessel 4 may have a pressure of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 psi. Further, according to some embodiments, the first (overhead) inert gas directed to and / or traveling through the upper interior of the reaction vessel 4 may have a pressure ranging from about any of the above pressure values ​​to about any other of the above pressure values.

[0115] As a non-limiting example, the second inert gas introduced into the lower interior of the reaction vessel 4 may have a pressure that is lower than the pressure of the first (overhead) inert gas directed into and / or traveling through the upper interior of the reaction vessel 4. For example, the second inert gas may have a pressure of about 1 psi to about 25 psi, so long as the pressure of the second inert gas is lower than the pressure of the first inert gas. In embodiments in which inert gas is introduced only into the lower interior of the reaction vessel (no inert gas is introduced into the upper interior of the reaction vessel), the inert gas may have a pressure of about 1 psi to about 25 psi.

[0116] In a non-limiting example, a first (overhead) inert gas directed to and / or traveling through the upper interior (e.g., headspace) of reaction vessel 4 may have a pressure of about 15 psi, and a second inert gas introduced to the lower part of reaction vessel 4 may have a lower pressure than the pressure of the first (overhead) inert gas directed to and / or traveling through the upper part of reaction vessel 4, e.g., a pressure of about 5 psi.

[0117] Thus, the process allows for the use of relatively low boiling deprotection agents at higher temperatures to speed up reaction times, while minimizing (reducing) the deleterious effects associated with using low boiling, volatile reactants. For example, the process can improve peptide purity even for longer peptide chains. Furthermore, the process can improve peptide purity even using pyrrolidine as the deprotection agent, even though significant evaporation is observed due to pyrrolidine's lower boiling point. This can also provide the benefit of faster deprotection reactions.

[0118] After the deprotection is complete, the flow of the inert gas is stopped and the deprotection agent is then vented, for example through opening 16.

[0119] The present disclosure also relates to a solid phase peptide synthesis process (e.g., a SPPS process comprising one or more deprotection steps according to the first embodiment and / or one or more deprotection steps according to the second embodiment) that includes one or more deprotection steps using an inert gas to purge the volatilized deprotection agent from the reaction vessel (e.g., to vent the volatilized deprotection agent from the headspace inside the reaction vessel) according to any of the embodiments of the deprotection step described in more detail herein. The solid phase peptide synthesis process of the present disclosure further includes coupling of amino acid(s) and / or washing, e.g., washing after the deprotection step and / or coupling step. The coupling and washing steps of solid phase peptide synthesis and systems for performing them are generally known in the art and therefore will not be described in detail herein.

[0120] As noted herein, conventional SPPS processes require multiple washing steps between the deprotection step and the conjugation step (e.g., after deprotection but before conjugation) to remove residual deprotection agent. In some embodiments of the present disclosure (e.g., SPPS processes including a deprotection step according to the first embodiment described herein), a washing solution (e.g., a solvent such as, but not limited to, dimethylformamide (DMF), methanol, and / or isopropanol) may be added to the vessel for the washing step after deprotection. The washing steps may be repeated (e.g., five times). The present disclosure is not limited to five washing steps, and fewer than five (e.g., one, two, three, or four) or more than five washing steps may be used.

[0121] However, washing steps may require the use of large amounts of solvent, which must be recovered and disposed of, etc. This can increase the cost of materials and the time of peptide synthesis, reduce efficiency, etc. Furthermore, as the length of the peptide increases, multiple washing steps may be less effective in preventing undesired reactions and reducing impurities, which may make it difficult to synthesize longer peptides with acceptable purity for downstream applications.

[0122] In some embodiments (e.g., the SPPS process including a deprotection step according to the second embodiment described herein), the present disclosure relates to an SPPS process including a deprotection step followed by a coupling step, which does not include a washing step after the deprotection step and before the associated coupling step. In other words, in the SPPS process of the present disclosure including a deprotection step(s) according to the second embodiment, one or more washing steps between the deprotection step and the associated coupling step (i.e., the coupling step immediately following the deprotection step) may be eliminated (e.g., all washing steps may be eliminated). This can provide benefits such as improved process efficiency, energy savings, reduced amount of solvent required in the SPPS process, reduced material costs, reduced solvent disposal issues, etc.

[0123] For example, an SPPS process of the present disclosure including a deprotection step(s) according to the second embodiment may include a series of deprotection-coupling cycles, where one or more (e.g. all) wash steps are eliminated (e.g. there are no wash steps) between at least one deprotection step and the coupling step of the deprotection-coupling cycles of the SPPS process. In another example, an SPPS process of the present disclosure including a deprotection step(s) according to the second embodiment may include a series of deprotection-coupling cycles, where one or more (e.g. all) wash steps are eliminated (e.g. there are no wash steps) between more than one deprotection step and the coupling step of the deprotection-coupling cycles, for example for half of the deprotection-coupling cycles of the SPPS process, for example for a majority of the deprotection-coupling cycles, and as another example for all of the deprotection-coupling cycles.

[0124] In yet another embodiment, the present disclosure relates to an SPPS process comprising a deprotection step(s) and a subsequent coupling step according to the second embodiment, the SPPS process comprising one or more washing steps (e.g., one, two, three, four, five, etc.) using a washing composition (e.g., a solvent) after the deprotection step and before the associated coupling step. However, in contrast to conventional washing steps, the washing step(s) of this embodiment uses a reduced amount of solvent. More specifically, the washing step(s) after the deprotection and before the coupling may comprise washing the interior of the reaction vessel one or more times (e.g., one, two, three, four, five, etc.) using a washing composition (e.g., a solvent) in an amount less than the total volume of the deprotection composition used in the deprotection step. For example, the washing step may comprise washing the interior of the reaction vessel one or more times (e.g., one, two, three, four, five, etc.) using a washing composition (e.g., a solvent) in an amount less than ½ the total volume of the deprotection composition used in the deprotection step. As another non-limiting example, the process may include washing the interior of the reaction vessel one or more times (e.g., 1, 2, 3, 4, 5, etc.) using a washing composition (e.g., solvent) in an amount less than ⅓ of the total volume of the deprotection composition used in the deprotection step. Additionally, the SPPS process may include a series of deprotection-coupling cycles, one or more of which (e.g., half, a majority, or all) include one or more washing steps (e.g., 1, 2, 3, 4, 5, etc. washing steps) between the deprotection step and the coupling step, and the washing step(s) uses a washing composition (e.g., solvent) in an amount less than the total volume of the deprotection composition used in the deprotection step (e.g., less than ½ of the total volume of the deprotection composition, as another example, less than ⅓ of the total volume of the deprotection composition). The volume of solvent used in each washing step and / or the total volume of solvent used in all washing steps in a given deprotection-coupling cycle (after deprotection and before the next coupling step) can be less than the total volume of the deprotection composition used in the deprotection step (e.g., less than ½ the total volume of the deprotection composition, as another example, less than ⅓ the total volume of the deprotection composition).

[0125] If used, the cleaning composition (cleaning solution) may include a solvent such as, but not limited to, dimethylformamide (DMF), methanol, and / or isopropanol.

[0126] If a washing step is used, in some embodiments, a washing liquid (e.g., solvent) can be introduced into the reaction vessel through a suitable opening into the upper interior of the reaction vessel, such as opening 10 in FIG. 1A, and / or using a different or the same spray head (e.g., spray head 220 in FIG. 1B) as used to introduce the inert gas into the reaction vessel during the deprotection step described herein. As a non-limiting example, as shown in FIG. 1B, the process may include providing (e.g., directing) a solvent from a solvent source (not shown in FIG. 1B), through flow path 218, opening 208, the inner interior space of spray head 220, and through opening 222 to the outer interior space 7 of reaction vessel 4. Also as shown generally in FIG. 1B, in some embodiments, spray head 220 can direct (e.g., spray) the solvent toward sidewall 6 of reaction vessel 4 through opening 222 at an angle (e.g., spray pattern) generally indicated by dashed line 224. This can facilitate washing the deprotection agent condensed on the sidewall downward toward the lower interior of reaction vessel 4.

[0127] If a washing step is included, the washing solution can be removed in a second draining step, after which the binding step can begin by known processes.

[0128] The solid phase peptide synthesis process of the present disclosure may more specifically include the steps of deprotecting a first amino acid (e.g., removing a protecting group of a first protected amino acid), which may be directly or indirectly coupled to a solid phase resin to form a deprotected amino acid, optionally washing the deprotected amino acid, coupling a second amino acid to the deprotected amino acid to form a peptide from the first and second amino acids, and repeating the deprotection, washing, and / or coupling steps to form a peptide comprising the first, second, and subsequent amino acids, one or more of the deprotection steps employing an inert gas purging step as described herein (e.g., according to the first and / or second embodiments of the deprotection step described herein).

[0129] In some embodiments, the solid phase peptide synthesis process includes the steps of deprotecting a first protected amino acid (e.g., by removing a protecting group of the first protected amino acid) to form a deprotected amino acid, washing the deprotected amino acid, coupling a second amino acid to the deprotected amino acid to form a peptide from the first and second amino acids, and repeating the steps of deprotecting, washing, and coupling to form a peptide comprising the first, second, and subsequent amino acids;

[0130] The deprotection and coupling steps are carried out in a reaction vessel (e.g., in the same reaction vessel, such as reaction vessel 4 described in more detail herein), and one or more of the deprotection steps are

[0131] heating the protected amino acid and the deprotection agent in a lower interior of the reaction vessel during the deprotection step, the heating volatilizing the deprotection agent into an upper interior (e.g., headspace) of the reaction vessel; and

[0132] During the heating step, the method may include directing a first inert gas into the upper interior of the reaction vessel through a first opening in the top of the reaction vessel, through the upper interior of the reaction vessel containing the volatilized deprotection agent (e.g., through the headspace), and from the upper interior of the reaction vessel through a second opening in the top of the reaction vessel to purge (vent) the volatilized deprotection agent from the interior of the reaction vessel.

[0133] In some embodiments, the solid phase peptide synthesis process may include deprotecting a first protected amino acid (e.g., removing a protecting group of the first protected amino acid) to form a deprotected amino acid, coupling a second amino acid to the deprotected amino acid to form a peptide from the first and second amino acids, and repeating the deprotecting and coupling steps to form a peptide comprising the first, second, and subsequent amino acids;

[0134] The deprotection and coupling steps are carried out in a reaction vessel (e.g., in the same reaction vessel, such as reaction vessel 4 described in more detail herein);

[0135] one or more of the deprotection steps employ a deprotection composition comprising a deprotecting agent in an amount of about 5 vol.% or less, based on the total volume of the deprotection composition, as described in more detail herein (e.g., according to the deprotection process of the second embodiment); and / or

[0136] One or more of the deprotection steps also employ a step of purging with an inert gas (e.g., venting the headspace) to remove evaporated deprotection agent from the upper interior of the reaction vessel (e.g., from the headspace), as described in more detail herein (e.g., according to the deprotection process of the second embodiment).

[0137] For example, a SPPS process including a deprotection step according to the second embodiment may include a step of directing an inert gas through the reaction vessel (e.g., through the upper interior of the reaction vessel containing the vaporized deprotection agent (through the headspace)) to remove the vaporized deprotection agent from the interior of the reaction vessel (e.g., from the upper interior of the reaction vessel, from the headspace).

[0138] More specifically, a SPPS process including a deprotection step according to the second embodiment may include directing (introducing, feeding, etc.) an inert gas into an upper interior of the reaction vessel through a first opening located at the top of the reaction vessel and through an upper interior (e.g., headspace) of the reaction vessel containing vaporized deprotection agent, and releasing (venting) the inert gas and vaporized deprotection agent from the upper interior (e.g., headspace) of the reaction vessel through a second opening also located at the top of the reaction vessel.

[0139] As another example, a SPPS process including a deprotection step according to the second embodiment may include directing (introducing, feeding, etc.) an inert gas into a lower interior of the reaction vessel through an opening located at the bottom of the reaction vessel and generally upwardly from the lower interior of the reaction vessel (e.g., generally upwardly through the reactants in the lower interior of the reaction vessel) into / through an upper interior (e.g., headspace) of the reaction vessel containing the vaporized deprotection agent, and releasing (venting) the inert gas and vaporized deprotection agent from the upper interior (e.g., headspace) of the reaction vessel through another opening located at the top of the reaction vessel.

[0140] As yet another example, a SPPS process including a deprotection step according to the second embodiment may include directing (introducing, feeding, etc.) an inert gas to both an upper interior of the reaction vessel and a lower interior of the reaction vessel, and venting (venting) the inert gas and vaporized deprotection agent from the upper interior (e.g., headspace) of the reaction vessel. More specifically, the deprotection step according to the second embodiment may include the steps of directing (introducing, feeding, etc.) an inert gas into the upper interior of the reaction vessel through a first opening located at the top of the reaction vessel and through the upper interior (e.g., headspace) of the reaction vessel containing the vaporized deprotection agent, and directing the inert gas into the lower interior of the reaction vessel through a second opening located at the bottom of the reaction vessel, and optionally upward from the lower interior of the reaction vessel (e.g., optionally generally upward through the reactants in the lower interior of the reaction vessel and into / through the upper interior (e.g., headspace) of the reaction vessel containing the vaporized deprotection agent), and releasing the inert gas and the vaporized deprotection agent from the upper interior of the reaction vessel through a third opening located at the top of the reaction vessel.

[0141] In the SPPS process including the deprotection step according to the second embodiment, the solid phase peptide synthesis process may not include a washing step between the successive deprotection and coupling steps. In other SPPS processes including the deprotection step according to the second embodiment, the solid phase peptide synthesis process may include one or more washing steps after the deprotection step and before the successive coupling steps, and the washing steps use a reduced amount of washing solution (e.g., solvent) as described in more detail herein (e.g., using a solvent in an amount less than the total volume of the deprotection composition used in the deprotection step, e.g., less than 1 / 2 the total volume of the deprotection composition, and as another example, less than 1 / 3 the total volume of the deprotection composition). For example, the solid phase peptide synthesis process may omit one or more (e.g., all) washing steps between one or more of the consecutive deprotection-coupling steps. As another example, the solid phase peptide synthesis process may include, for one or more of the sequential deprotection-coupling steps, after deprotection and prior to the associated coupling step, a step of washing the deprotected amino acids with a reduced amount of wash solution (e.g., solvent) as described in more detail herein (e.g., using a solvent in an amount less than the total volume of the deprotection composition used in the deprotection step, for example less than ½ the total volume of the deprotection composition, and as another example less than ⅓ the total volume of the deprotection composition).

[0142] The solid phase peptide synthesis process may further include, prior to conjugation, activating the chemical group(s) of the second amino acid (and subsequent amino acid(s)) using processes and agents known in the art to prepare the second (and subsequent) amino acid(s) for conjugation with the first (and sequential) amino acid(s).

[0143] Prior to the coupling step, an amino acid activating agent may be used to activate the amino acid (e.g., convert the acid group of the amino acid to an activated form). Any suitable amino acid activating agent may be used. Examples of amino acid activating agents include, without limitation, carbodiimides and / or onium salt activating agents. Amino acid activating agents, in some embodiments, include carbodiimides, such as, but not limited to, N,N'-diisopropylcarbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and combinations thereof. In certain embodiments, amino acid activators include onium activators such as, but not limited to, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), and 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholino)]uronium hexafluorophosphate (COMU), and combinations thereof.

[0144] Additionally, in exemplary embodiments, the solid phase peptide synthesis process may include applying microwave energy during one or more of the solid phase peptide synthesis steps, for example during the deprotection and / or coupling steps.

[0145] In an exemplary embodiment, the solid phase peptide synthesis process may further comprise the step of cleaving the peptide from the solid phase resin after the deprotection, washing, and / or coupling steps.

[0146] Those skilled in the art will understand how to join or bond amino acids to form a chain. Processes and agents for cleaving peptides from solid-phase resins are also well known in the art. Therefore, a detailed discussion of the art-known processes for joining amino acids to form peptides and / or for cleaving peptides from solid-phase resins is not provided.

[0147] The present disclosure also relates to a system for solid phase peptide synthesis. Figure 2A is a schematic flow diagram showing selected portions of an exemplary solid phase peptide synthesis system according to an embodiment of the present disclosure. Figure 2B is a schematic flow diagram showing selected portions of another exemplary peptide synthesis system according to another embodiment of the present disclosure.

[0148] Generally, elements shown in both Figures 1A and 2A will have the same reference numbers. Similarly, elements shown in both Figures 1B and 2B will generally have the same reference numbers. Also, unless otherwise indicated, elements shown in both Figures 2A and 2B will have the same reference numbers.

[0149] 2A and 2B is generally designated 2. Peptide synthesis system 2 includes a reaction vessel 4 as discussed herein. Peptide synthesis system 2 also includes a number of reagent containers located upstream of and in fluid communication with reaction vessel 4.

[0150] 2A and 2B, system 2 may include multiple solid support containers 50a, 50b, and 50c in fluid communication with a flow path 52 that fluidly connects the solid support containers to reaction vessel 4 for delivering solid supports (e.g., solid resins having protected amino acids attached thereto) from the solid support container(s) to reaction vessel 4. Flow paths 51a, 51b, and 51c fluidly connect solid support containers 50a, 50b, and 50c, respectively, to flow path 52.

[0151] In certain embodiments, the flow path 52 may be in direct fluid communication with the reaction vessel 4, for example, via opening 12 or 204 in FIG. 1A or FIG. 1B, respectively. In certain embodiments, as shown generally in FIG. 2A, the system may include a rotary valve 140 rotatable between multiple positions (e.g., two positions) to fluidly couple the reaction vessel 4 to the solid support containers 50a, 50b, and 50c through opening 12, flow path 22, and flow path 52. In certain other embodiments, as shown generally in FIG. 2B, the rotary valve 140 is rotatable between multiple positions (e.g., two or more positions) to fluidly couple the reaction vessel 4 to the solid support containers 50a, 50b, and 50c through opening 204, flow path 214, and flow path 52.

[0152] As another example, in certain embodiments, as shown generally in Figures 2A and 2B, system 2 may include a plurality of amino acid containers 60a, 60b, and 60c in fluid communication with a channel 62 (Figure 2A) or a channel 212 (Figure 2B) that fluidly couples the amino acid containers to reaction vessel 4 for delivery of protected amino acids from the amino acid container(s) to reaction vessel 4. Channels 61a, 61b, and 61c fluidly couple amino acid containers 60a, 60b, and 60c to channel 62 (Figure 2A) or channel 212 (Figure 2B), respectively. In some embodiments, channel 62 and / or channel 212 may be in direct fluid communication with reaction vessel 4, e.g., via openings 10 or 202 in Figures 1A or 1B, respectively. In some embodiments, the system may include one or more additional valves and / or channels, as shown generally in Figures 2A and 2B and described in more detail below.

[0153] As another example, as shown generally in Figures 2A and 2B, system 2 may include a deprotection agent container 70 in fluid communication with a channel 72 fluidly connecting the deprotection agent container and reaction vessel 4 for delivering a deprotection agent from the deprotection agent container to reaction vessel 4. In some embodiments, channel 72 may be in direct fluid communication with reaction vessel 4, for example, via opening 16 in Figures 1A and 1B. In some embodiments, the system may include one or more additional valves and / or channels, as shown generally in Figures 2A and 2B and described in more detail below.

[0154] As yet another example, as shown generally in Figures 2A and 2B, system 2 may include a solvent reservoir 80 in fluid communication with a flow path 82 fluidly connecting the solvent reservoir and reaction vessel 4 for delivering solvent from the solvent reservoir to reaction vessel 4. In some embodiments, flow path 82 may be in direct fluid communication with reaction vessel 4, for example, via opening 10 or 208 in Figures 1A or 1B, respectively. In some embodiments, the system may include one or more additional valves and / or flow paths, as shown generally in Figures 2A and 2B and described in more detail below. Figure 2B also shows generally an embodiment in which solvent may be introduced into reaction vessel 4 using at least some of the same flow paths used to introduce inert gas, for example, via flow path 218, opening 208, spray head 220, and multiple openings 222, as described in more detail herein.

[0155] As yet another example, as shown generally in Figures 2A and 2B, system 2 may include an additional reagent container 90, which may be an activator container in fluid communication with a channel 92 (Figure 2A) or channel 210 (Figure 2B) fluidly connecting the additional reagent container and reaction vessel 4 for delivering an additional reagent, such as an activator, from the additional reagent container to reaction vessel 4. In some embodiments, channel 92 or 210 may be in direct fluid communication with reaction vessel 4, for example via opening 10 or 200 in Figures 1A and 1B, respectively. In some embodiments, system 2 may include one or more additional valves and / or channels, as shown generally in Figures 2A and 2B and described in more detail below.

[0156] Those skilled in the art will recognize that the number of reaction vessels, solid support reservoirs, amino acid reservoirs, deprotection agent reservoirs, solvent reservoirs, and / or other reagent reservoirs, and associated flow paths, as well as the manner in which these elements are connected, may vary and are not limited to their depictions in Figures 2A and 2B. Those skilled in the art will also appreciate that a peptide synthesis system may include various subsystems associated with the above reservoirs, flow paths, and / or reaction vessel(s), including, for example, flow paths, valves, filters, gauges, monitors, controllers, etc., to direct the flow of materials into and / or out of the reservoirs and / or reaction vessel(s) at appropriate stages of the solid phase peptide synthesis process. Such subsystems are well known in the art and will not be described in detail herein.

[0157] System 2 also involves a heat source (not shown), such as a microwave source for heating reaction vessel 4, and associated elements, such as a microwave guide and / or microwave cavity, as described herein. Heat sources, including microwave heat sources, and associated elements, such as microwave guides and / or microwave cavities, and their use in processes and systems for solid phase peptide synthesis are also well known in the art and will not be described in greater detail herein.

[0158] System 2 is shown diagrammatically as operating in an amino acid deprotection step of solid phase peptide synthesis, e.g., as described herein with reference to Figures 1A and 1B. In this operational state, reactants including a protected amino acid and a deprotection agent have already been delivered to (are already present in) reaction vessel 4.

[0159] The protected amino acid is bound to a solid support. The protected amino acid may be directly bound to a solid support delivered into reaction vessel 4 via flow channel 52 from, for example, one or more of solid support containers 50a, 50b, and 50c. Alternatively, the protected amino acid may be indirectly bound to the solid support (e.g., bound to another amino acid or peptide chain, which is bound to the solid support).

[0160] The system 2 further includes an inert gas source 100 located upstream of and in fluid communication with the reaction vessel 4. In certain embodiments, as shown generally in Figures 2A and 2B, a flow path 102 fluidly connects the inert gas source 100 to the reaction vessel 4 and delivers inert gas provided by the inert gas source 100 to an upper interior of the reaction vessel 4 via an opening 10 (Figure 1A) or an opening 208 (Figure 1B) as described herein. In certain embodiments, the system 2 includes a valve 104 in fluid communication with the flow path 102 located between the inert gas source 100 and the opening 10 (Figure 1A) or the opening 208 (Figure 1B), the valve 104 having an open position and a closed position with respect to the flow path 102.

[0161] Valve 104 is shown in an open position relative to flow path 102 in Figures 2A and 2B. In the open position, valve 104 allows flow of pressurized inert gas from inert gas source 100 through flow path 102, flow path 20 (Figure 1A) or flow path 218 (Figure 1B), and through opening 10 (Figure 1A) or opening 208 and spray head 220 (Figure 1B) to the interior upper portion of reaction vessel 4 (e.g., to the headspace of the reaction vessel). In this manner, the system can continuously and / or intermittently direct an overhead source of pressurized inert gas to the interior upper portion of the reaction vessel during heating and / or deprotection steps to purge (vent, release) volatilized deprotection agent from the headspace of the reactor according to the processes described herein.

[0162] In contrast, when valve 104 is in a closed position relative to flow path 102, valve 104 can prevent the flow of pressurized inert gas from the inert gas source through flow path 102, flow path 20 (FIG. 1A) or flow path 218 (FIG. 1B) and opening 10 (FIG. 1A) or opening 208 and spray head 220 (FIG. 1B).

[0163] The system may include a flow path 106 fluidly connecting the inert gas source 100 and an opening 16 at the bottom of the reaction vessel 4. The system may include a valve 108 in fluid communication with the flow path 106 located between the inert gas source 100 and the opening 16, the valve 108 having an open position and a closed position with respect to the flow path 106.

[0164] In an open position relative to the flow path 106, the valve 108 allows the flow of pressurized gas from the inert gas source 100 through the flow path 106, the flow path 26, and the opening 16 to the lower interior of the reaction vessel 4. As described herein, in a deprotection process, the pressurized inert gas can be directed to the lower part of the reaction vessel to agitate (e.g., stir, bubble) the reactants and / or flush vaporized deprotection agent from the headspace of the reaction vessel. A second valve 108 in a closed position relative to the flow path 106 prevents the flow of pressurized inert gas from the inert gas source 100 through the flow path 106, the flow path 26, and the opening 16 to the lower interior of the reaction vessel 4.

[0165] In one particular embodiment, flow paths 102 and 106 may be fluidly connected to a single inert gas source 100 via a pressure regulator, designated 110 in Figures 2A and 2B. Alternatively, flow paths 102 and 106 may fluidly connect the reaction vessel to at least two different inert gas sources.

[0166] When present, the pressure regulator 110 may be located downstream from the inert gas source 100 and upstream from the valves 104 and 108. In these embodiments, the pressure source 100 directs inert gas to the pressure regulator 110, which supplies pressurized inert gas ("high pressure" inert gas) to the flow path 102 having a higher pressure than the inert gas ("low pressure" inert gas) supplied to the flow path 106. For example, and without limitation, the pressure regulator 110 may supply a "high pressure" inert gas to the flow path 102 having a pressure of about 1 psi to about 25 psi. The pressure regulator 110 may also supply a "low pressure" inert gas having a lower pressure than the pressure of the "high pressure" inert gas supplied to the flow path 102.

[0167] Pressure regulators are also well known in the art, and one of ordinary skill in the art would understand how to use them in system 2 to provide high and low pressure inert gas as discussed herein.

[0168] As also shown generally in Figures 2A and 2B (as well as Figures 1A and 1B), in an exemplary embodiment, the system may include a flow path 24 (Figures 1A and 2A) or a flow path 216 (Figures 1B and 2B) located downstream from opening 14 (Figure 1A) or opening 206 (Figure 1B) of reaction vessel 4. Flow paths 24 and 216 may function as gaseous waste flow paths and allow for the purging of gaseous waste (e.g., volatilized deprotection agent, inert gas, etc.) from the upper interior (headspace) of reaction vessel 4 during the deprotection processes described herein.

[0169] In certain embodiments, the system includes a valve 120 in fluid communication with the flow path 24 or flow path 216. The valve 120 has an open and closed position relative to the flow path 24 or flow path 216. The valve 120 in the open position allows the flow of gas from the upper interior (head space) of the reaction vessel 4 through the opening 14 or opening 206 and the flow path 24 or flow path 216 to a waste collection zone, such as a vent and / or waste receptacle (not shown), and allows for purging / releasing of gas from the reaction vessel 4. The valve 120 in the closed position prevents the flow of gas from the upper interior (head space) of the reaction vessel through the opening 14 or opening 206 to the outside.

[0170] 2A and 2B illustrate a particular embodiment of the system in an operational state in which both valves 104 and 120 are in an open position relative to flow path 102, flow path 20 or 218, and flow path 24 or 216, respectively. This corresponds to a position that can be used during the (heated) deprotection process described herein. Valves 104 and 120 simultaneously in an open position relative to flow path 102, flow path 20 or 218, and flow path 24 or 216, respectively, allow an overhead supply of pressurized inert gas to flow continuously and / or intermittently through reaction vessel 4 (e.g., into vessel 4 through opening 10 in the vessel headspace and out of vessel 4 through another opening 14, or as another example, into vessel 4 through opening 208, spray head 220, and opening 222 toward sidewall 6, into the vessel headspace, and out of vessel 4 through another opening 206) to purge (vent) volatilized reactants present in the headspace during the heated deprotection step.

[0171] In certain embodiments, the system 2 may include a valve 122 in series with the valve 104, and a flow path 124 positioned between and fluidly connecting the valves 104 and 122. The valve 122 is in fluid communication with the flow path 102 and has an open position and a closed position relative to the flow path 102. As shown in Figures 2A and 2B, when the valve 122 is present and the valves 122 and 104 are in an open position relative to the flow path 102, the inert gas source 100, the pressure regulator 110, the flow path 102, the valve 104, the flow path 124, the valve 122, the flow path 20 (Figure 2A) or flow path 218 (Figure 2B), the opening 10 (Figures 1A / 2A) or opening 208, and the spray head 220 (Figures 2A / 2B), and the reaction vessel 4 may be in fluid communication.

[0172] In certain embodiments, the valve 104 may be a rotary valve that can rotate between multiple positions to fluidly couple a selected one of the multiple flow paths with the reaction vessel 4. As a non-limiting example, FIG. 2A illustrates a rotary valve 104 that can rotate between four positions to fluidly communicate with the flow paths 102, 62, 82, or 92 depending on the open or closed position of the valve. For example, FIG. 2A illustrates a rotary valve 104 that is in an open position with respect to the flow path 102, but in a closed position with respect to the flow paths 62, 82, and 92. As another non-limiting example, FIG. 2B illustrates a rotary valve 104 that can rotate between two positions to fluidly communicate with the flow paths 102 or 82 depending on the open or closed position of the valve. For example, FIG. 2B illustrates a rotary valve 104 that is in an open position with respect to the flow path 102, but in a closed position with respect to the flow path 82. The open / closed positions of the rotary valve 104 with respect to the various flow paths may be selected depending on the stage of the peptide synthesis process, the reactants to be delivered into the reaction vessel 4, etc. The rotary valve 104 (and the other valves discussed herein) may be operated as known in the art.

[0173] Other valves in the system may also be rotary valves. For example, as noted herein, in certain embodiments, system 2 may include a flow path 124 positioned between and fluidly connecting valve 104 and valve 122. In this embodiment, as shown generally in Figures 2A and 2B, valve 122 in series with valve 104 may rotate between multiple positions (e.g., valve 122 between two positions in Figures 2A and 2B and valve 104 between four positions generally shown in Figure 2A and two positions generally shown in Figure 2B) to fluidly connect inert gas source 100 and reaction vessel 4, for example, via flow path 102, flow path 124, flow path 20 or flow path 218, and opening 10 or opening 208. Alternatively, as shown in Figure 2A, valve 122 in series with valve 104 can be rotated between multiple positions (e.g., two positions and four positions, respectively) to fluidly couple, for example, one or more of amino acid containers 60a, 60b, and 60c to reaction vessel 4 via flow channel 62, flow channel 124, flow channel 20, and opening 10, solvent container 80 to reaction vessel 4 via flow channel 82, flow channel 124, flow channel 20, and opening 10, or reagent container 90 to reaction vessel 4 via flow channel 92, flow channel 124, flow channel 20, and opening 10. Also, in some embodiments, as shown in Figure 2B, valve 122 in series with valve 104 can be rotated between multiple positions (e.g., two positions) to fluidly couple, for example, solvent container 80 to reaction vessel 4 via flow channel 82, flow channel 124, flow channel 218, and opening 208, as well as spray head 222.

[0174] As another example, in certain embodiments, as shown diagrammatically in Figures 2A and 2B, valve 108 may be a rotary valve that rotates between a number of positions (e.g., three positions) to fluidly connect reaction vessel 4 with deprotection agent reservoir 70, inert gas source 100, or waste reservoir 130, respectively, through opening 16, flow path 26, and flow path 72, flow path 106, or flow path 132, depending on the position of valve 108. For example, Figures 2A and 2B diagrammatically show rotary valve 108 in an open position with respect to flow path 106, but in a closed position with respect to flow paths 72 and 132. This may be the position of rotary valve 108 during the heating and / or deprotection processes described herein, in which low pressure inert gas is directed (bubbled) into the bottom of reaction vessel 4 to agitate reactants and / or flush vaporized deprotection agent from the headspace of the reaction vessel.

[0175] As another example, as discussed herein, in certain embodiments, as also shown generally in FIG. 2A, the system may include a rotary valve 140 that can rotate between multiple positions (e.g., two positions) to fluidly couple reaction vessel 4 to solid support containers 50a, 50b, and 50c through opening 12, channel 22, and channel 52. Alternatively, rotary valve 140 can rotate between multiple positions (e.g., two positions) to fluidly couple reaction vessel 4 to opening 12, channel 22, and channel 152, which fluidly couples channel 152 to multiple product containers 150a, 150b, and 150c, respectively. This allows for the passage of products (e.g., peptides and / or peptides bound to solid supports) from reaction vessel 4 to containers 150a, 150b, and 150c. Again, one of ordinary skill in the art will recognize that the number of product containers 150a, 150b, and 150c and corresponding flow channels 151a, 151b, and 151c may vary and are not limited to the number shown in FIG. 2A.

[0176] As another example, as discussed herein, in certain embodiments, as also shown generally in FIG. 2B, the system may include a rotary valve 140 that can rotate between multiple positions (e.g., two positions) to fluidly couple reaction vessel 4 to solid support containers 50a, 50b, and 50c through opening 204, channel 214, and channel 52. Alternatively, rotary valve 140 can rotate between multiple positions (e.g., two positions) to fluidly couple reaction vessel 4 to solid support containers 50a, 50b, and 50c through opening 204, channel 214, and channel 152, again in fluid communication with multiple channels 151a, 151b, and 151c, which in turn fluidly couple channel 152 to multiple product containers 150a, 150b, and 150c, respectively. Again, this allows for the passage of products (e.g., peptides and / or peptides bound to solid supports) from reaction vessel 4 to containers 150a, 150b, and 150c. Again, those skilled in the art will recognize that the number of product containers 150a, 150b, and 150c and corresponding flow channels 151a, 151b, and 151c may vary and are not limited to the number shown in FIG. 2B.

[0177] The peptide synthesis system 2 may include one or more flow paths, vents, containers, valves, controllers, etc., for example, removal of waste products (e.g., excess reactants, solvents, etc.) from the peptide synthesis system. The waste products may be in gas, liquid, and / or solid form, and one of skill in the art will recognize suitable types of flow paths and containers for removal of waste products from the peptide synthesis system. For example, in certain embodiments, as discussed herein, the waste container 130 may be fluidly connected to the reaction vessel 4 via the flow path 132 and the rotary valve 108 when in an appropriate open position to allow the passage of waste products from the reaction vessel 4 to the waste container 130. As another example, in certain embodiments, as discussed herein, the valve 120 in an open position allows the flow of gas from the upper interior (headspace) of the reaction vessel 4 through the opening 14 or opening 206 and the flow path 24 or flow path 216 to a waste collection zone, e.g., a vent and / or a waste container (not shown), to allow purging / releasing of gas from the reaction vessel 4.

[0178] 2A and 2B show an exemplary system for the delivery of solvents, reactants (amino acids, solid phase resins, deprotectants, activators, etc.), gases, etc. from their respective sources to reaction vessel 4, and for the further delivery of products and by-products (peptides, gaseous, liquid, and / or solid waste, etc.) from reaction vessel 4 to their respective destinations. It will be understood that the particular flow paths and valve locations are illustrative rather than limiting of the present disclosure.

[0179] To reiterate at least in part from above, a peptide synthesis system typically includes at least one controller operably associated with, for example, a number of electrical components of the system (e.g., microwave sources, sensors, and solenoids and / or other motor-driven valves). The at least one controller may include one or more computers, computer data storage devices, programmable logic devices (PLDs), and / or application specific integrated circuits (ASICs). A suitable computer may include one or more of each of a central processing unit or processor, computer hardware integrated circuits or memory, a user interface, peripheral or device interfaces for interfacing with other electrical components of the system, and / or any other suitable mechanisms. Each of the controller(s) may also communicate with the electrical components of the system by suitable signal communication paths. Figures 2A and 2B show schematic diagrams of representative signal communication paths associated with the controllers, each of which is numbered. * 2 (signal communication path) and * 1 (controller). The process of the present disclosure includes at least one controller * 1. may be controlled (e.g., at least partially controlled) in response to the execution of a computer-based algorithm operably associated with the

[0180] Since solid-phase peptide synthesis processes, including batch-based processes, are known, this disclosure does not provide detailed information thereon. See, for example, the pioneering work of RB Merrifield (1963) "Solid Phase Peptide Synthesis I, The Synthesis of a Tetrapeptide," J. Am. Chem. Soc. 85 (14), 2149-2154). Accordingly, a detailed discussion of solid-phase peptide synthesis processes and systems is not provided.

[0181] Suitable systems and processes for performing solid phase peptide synthesis, including batch-based processes, are also known. Exemplary systems for performing solid phase peptide synthesis include, for example, the LIBERTY line of instruments commercially available from CEM Corporation of Matthews NC. Exemplary United States patents dealing with the subject of solid phase peptide synthesis include, without limitation, U.S. Patent Nos. 7,393,920, 7,550,560, 7,563,865, 7,939,628, 7,902,488, 7,582,728, 8,153,761, 8,058,393, 8,426,560, 8,846,862, 9,211,522, 9,669,380, 10,052,607, 10,308,677, 10,125,163, 10,858,390, and 10,239,914, the contents of each of which are incorporated herein by reference in their entirety.

[0182] The deprotection and / or SPPS processes of the present disclosure may be used in combination with SPPS processes that do not include (or exclude) washes after each coupling step and / or add the deprotecting base directly to the coupling solution without draining after the coupling step (e.g., SPPS processes in which the protected amino acid includes an Fmoc protecting group), such as those disclosed in U.S. Patent Nos. 10,308,677, 10,125,163, 10,858,390, and 10,239,914. Such SPPS processes may be generally referred to as "high efficiency SPPS (HE-SPPS)."

[0183] The present invention will now be described in more detail with reference to the following examples, it being understood that these examples are provided for illustrative purposes only and should not be construed as limiting the present invention in any manner. EXAMPLES

[0184] Example 1 Synthesis of thymosin with headspace cleaning during deprotection. The peptide thymosin SDAAVDTSSEITTKDLKEKKEVVEEAEN-NH2 is synthesized using solid-phase peptide synthesis on an automated microwave peptide synthesizer Liberty PRIME commercially available from CEM Corporation (Matthews, NC) (synthesis scale 0.05 mmol). Rink Amide ProTide LL (0.19 mmol / g substitution) is used as the solid-phase resin support.

[0185] Deprotection is carried out by adding 0.75 mL of deprotection reagent containing 40% pyrrolidine in dimethylformamide (DMF) to the undrained post-coupling mixture. The microwave power is adjusted to give a deprotection temperature of 110° C., and the deprotection step is carried out at 110° C. for 50 seconds. After each deprotection step, the amino acid is washed three times with 5 mL of DMF.

[0186] During deprotection, according to the embodiments of the disclosure described herein, a stream of pressurized nitrogen gas at a pressure of about 15 psi (+ / - 3 psi) is continuously directed through the headspace of the reaction vessel to continuously purge the headspace gas from the reaction vessel.

[0187] The coupling reaction is carried out in the presence of a 10-fold molar excess of Fmoc-protected amino acid (AA) and activator / activator base diisopropylcarbodiimide (DIC) and ethyl(hydroxyimino)cyanoacetate (Oxyma), specifically AA / DIC / Oxyma (10:20:10) in 3.5 mL of DMF. The coupling reaction is carried out under microwave conditions selected to provide a 30 second wait and 90 seconds at 105°C.

[0188] After the synthesis of the peptide is complete, the peptide is cleaved from the solid phase using trifluoroacetic acid (TFA), triisopropyle silane (TIS), water (HO), and dioxa-1,8-octane-dithiol (DODT), specifically 5 mL of TFA / TIS / HO / DODT (92.5:2.5:2.5:2.5), at 38° C. for 30 minutes using the RAZOR cleavage system commercially available from CEM Corporation.

[0189] Peptides are analyzed using a Thermo Scientific Vanquish UPLC coupled to an Extractive Plus Orbitrap mass spectrometer using acetonitrile / water with 0.1% TFA as the solvent system and a C8 column (1.7 μm, 2.1×100 mm) or a Waters UPLC ACQUITY H-Class with a 3100 Single Quad mass spectrometer using acetonitrile / water with 0.1% TFA as the solvent system and a C8 column (1.7 μm, 2.1×100 mm). Comparative Example 1 Synthesis of thymosin without headspace cleaning during deprotection.

[0190] The peptide thymosin is synthesized under the same conditions as described in Example 1, except that the deprotection step did not involve continuously directing a stream of pressurized nitrogen gas through the headspace of the reaction vessel (there is no purging or cleaning of the headspace during deprotection).

[0191] The resulting peptides are analyzed, also as described in Example 1.

[0192] Figures 3A and 3B are UPLC chromatographs of thymosins of Example 1 and Comparative Example 1, respectively, demonstrating the increase in peptide purity using headspace purging (headspace cleaning) during the deprotection step according to the present disclosure. For example, as shown in Figure 3A, thymosins produced using headspace purging (headspace cleaning) during the deprotection step according to the present disclosure exhibit an improved purity of 76%. In contrast, as shown in Figure 3B, thymosins produced without headspace purging (headspace cleaning) during deprotection exhibit a reduced purity of 69%. Example 2 Synthesis of proinsulin with headspace cleaning during deprotection

[0193] The peptide proinsulin FVNQHLKGSH LVEALYLVKG ERGFFYTPKT RREAEDLQVG QVELGGGPGA GSLQPLALEG SLQKRGIVEQ KKTSIKSLYQ LENYKN (MW=9544; 1909 (+5 ion), 1591 (+6 ion), 1364 (+7 ion), 1194 (+8 ion), 1061 (+9 ion), 955 (+10 ion), 868 (+11 ion), 796 (+12 ion), 735 (+13 ion), 682 (+14 ion)) is synthesized under the same conditions described in Example 1, including continuously directing a stream of pressurized nitrogen gas through the headspace of the reaction vessel during deprotection (there is purging or cleaning of the headspace during deprotection).

[0194] The resulting peptides are analyzed, also as described in Example 1. Figures 4A and 4B are the UPLC chromatograph and mass spectrum, respectively, of the proinsulin of Example 2. Example 3 Synthesis of HIV-1 protease with headspace cleaning during deprotection

[0195] Peptide HIV-1 protease PQVTLWQRPI VTIKIGGQLK EALLDTGADD TVLEEMSLPG KWKPKMIGGI GGFIKVRQYD QVSIEIKGHK AIGTVLIGPT PVNIIGRNLL TQLGKTLNF (MW=10775; 1540 (+7 ion), 1347 (+8 ion), 1198 (+9 ion), 1078 (+10 ion), 980 (+11 ion), 898 (+12 ion), 829 (+13 ion), 770 (+14 ion)) is synthesized under the same conditions described in Example 1, including continuously directing a stream of pressurized nitrogen gas through the headspace of the reaction vessel during deprotection (there is purging or cleaning of the headspace during deprotection).

[0196] The resulting peptides are analyzed, also as described in Example 1. Figures 5A and 5B are the UPLC chromatograph and mass spectrum, respectively, of the HIV-1 protease of Example 3. Example 4 Synthesis of Barstar with Headspace Cleaning During Deprotection

[0197] The peptide Barstar KKAVINGEQI RSISDLHQTL KKELALPEYY GENLDALWDK LTGWVEYPLV LEWRQFEQSK QLTENGAESV LQVFREAKAE GKDITIILS (MW=10261; 1711 (+6 ion), 1466 (+7 ion), 1283 (+8 ion), 1141 (+9 ion), 1027 (+10 ion), 933 (+11 ion), 855 (+12 ion)) is synthesized under the same conditions described in Example 1, including continuously directing a stream of pressurized nitrogen gas through the headspace of the reaction vessel during deprotection (there is purging or cleaning of the headspace during deprotection).

[0198] The resulting peptides are analyzed, also as described in Example 1. Figures 6A and 6B are the UPLC chromatograph and mass spectrum, respectively, of the Barstar of Example 4. Example 5 Synthesis of MDM2 with headspace cleaning during deprotection

[0199] Peptide MDM2 MHHHHHHGSM KNTNMSVPTD GAVTTSQIPA SEQETLVRPK PLLLKLLKSV GAQKDTYTMK EVLFYLGQYI MTKRLYDEKQ QHIVYKSNDL LGDLFGVPSF SVKEHRKIYT MIYRNLVVVN QQESSDS (MHHHHHHGSM KNTNMSVPTD GAVTTSQIPA SEQETLVRPK PLLLKLLKSV GAQKDTYTMK EVLFYLGQYI MTKRLYDEKQ QHIVYKSNDL LGDLFGVPSF SVKEHRKIYT MIYRNLVVVN QQESSDS (MW=14607; 1218 (+12 ion), 1125 (+13 ion), 1044 (+14 ion), 975 (+15 ion), 913 (+16 ion), 860 (+17 ion), 813 (+18 ion)) is synthesized under the same conditions described in Example 1, including continuously directing a stream of pressurized nitrogen gas through the headspace of the reaction vessel during deprotection (with purging or cleaning of the headspace during deprotection).

[0200] The resulting peptides are analyzed, also as described in Example 1. Figures 7A and 7B are the UPLC chromatograph and mass spectrum, respectively, of MDM2 from Example 5. Example 6 Analysis of the one-pot synthesis of JR10-mer using low base concentrations, with and without post-deprotection washes, and with and without headspace venting.

[0201] Examples 1-5 above demonstrate that a high temperature deprotection step with inert gas (nitrogen) headspace bleed by microwave SPPS up to 110° C. can result in high purity even for long and difficult sequences. Examples 6 and 7 demonstrate that the use of even small amounts of deprotecting base can result in essentially complete deprotection and removal of protecting groups (e.g., Fmoc protecting groups), leaving only residual base that may be small enough to minimize challenges for the next coupling step.

[0202] The JR10-mer is synthesized using solid phase peptide synthesis using a commercially available automated microwave peptide synthesizer (e.g., the Liberty line of microwave peptide synthesizers, e.g., Liberty 2.0, commercially available from CEM Corporation, Matthews, NC). PEG-PS resin (e.g., Rink Amide ProTide Resin LL, commercially available from CEM Corporation) is used as the solid phase resin support, and the coupling reaction is carried out in the presence of Fmoc-protected amino acids (AA).

[0203] The deprotection reaction is carried out by adding pyrrolidine / dimethylformamide (DMF) deprotection reagent (composition) to the undrained post-coupling mixture. The pyrrolidine concentration (volume percent of pyrrolidine based on the total volume of the deprotection reagent, including pyrrolidine and DMF) is listed in Table 1 below. The microwave power is adjusted to provide the deprotection temperature and deprotection reaction time, also listed in Table 1 below.

[0204] Table 1 further indicates whether a post-deprotection wash and / or headspace bleed is used. For samples in Table 1 labeled with "headspace bleed" as "ON", a stream of nitrogen gas is directed through the headspace of the reaction vessel (e.g., a stream of pressurized nitrogen gas is directed through an entry port as shown in Figures 1A and 1B, through the headspace into the reaction vessel, and out of the reaction vessel through an exit port such as a vent port as shown in Figures 1A and 1B) to purge the headspace gas from the reaction vessel in accordance with the embodiments of the disclosure described herein. For samples in Table 1 labeled with "headspace bleed" as "OFF", headspace bleed as described herein is not used. For samples in Table 1 where a "post-protection wash" is used, the wash step includes two washes using 4 mL of DMF after each deprotection step.

[0205] After the synthesis of the JR10-mer is complete, the JR10-mer is cleaved from the solid phase and the crude purity of the resulting JR10-mer is analyzed. These results are also reported in Table 1 below. [Table 1]

[0206] Results for the synthesis of JR peptides indicate that high purity results can be obtained even without washing when headspace bleeds are used as described herein for each deprotection step. For example, without being bound by any explanation or theory and without limiting the scope of the present invention, it is presently believed that directing an inert gas (nitrogen gas) through an entry port (e.g., as shown in Figures 1A and 1B), through the headspace into the reaction vessel, and out of the reaction vessel through an exit port (e.g., a vent port as shown in Figures 1A and 1B) can result in both a higher gas exchange rate above the deprotection solution and a top-to-bottom directed flow that pushes the condensate back into the reaction vessel where it is reheated.

[0207] Furthermore, again without being bound by any explanation or theory and without limiting the scope of the invention, it is presently believed that Example 6 demonstrates that pyrrolidine base, which has a lower boiling point (87° C.) compared to piperidine (106° C.), can be significantly evaporated in the Fmoc removal step, and that even use of smaller amounts (as low as 2 vol%) can result in essentially complete deprotection and removal of the Fmoc group, leaving only residual base that may be sufficiently small to minimize challenges for the next coupling step. It is also presently believed that this is the first SPPS process demonstrated that eliminates all washing steps between standard cycles, and that this SPPS process generates only 4.25 mL of total waste for each standard amino acid cycle at a typical 0.1 mmol research scale. Example 7 No post-deprotection wash, headspace bleed, and use of a protection composition having a low base concentration 1-42 Analysis of the one-pot synthesis of sequences of β-amyloid and liraglutide

[0208] Two other notoriously difficult sequences, viz. 1-42 The sequences of β-amyloid and liraglutide are considered below. These sequences are synthesized using solid-phase peptide synthesis using a commercially available automated microwave peptide synthesizer (e.g., Liberty line microwave peptide synthesizer, e.g., Liberty 2.0, commercially available from CEM Corporation, Matthews, NC). Fmoc-Gly-Wang-Protide or Fmoc-Ala-Wang-Protide resin is used as the solid-phase resin support, and the coupling reaction is carried out in the presence of Fmoc-protected amino acids (AA).

[0209] The deprotection reaction is carried out by adding pyrrolidine / dimethylformamide (DMF) deprotection reagent (composition) to the undrained post-coupling mixture. The pyrrolidine concentration (volume percentage of pyrrolidine based on the total volume of the deprotection reagent, including pyrrolidine and DMF) is listed in Table 2 below. Based on the results of Example 6, a pyrrolidine concentration of 3 vol% is selected as the median process utilization for the synthesis of these sequences. The microwave power is adjusted to provide the deprotection temperature and deprotection reaction time, also listed in Table 2 below.

[0210] Table 2 shows that no post-deprotection wash is used, but headspace bleed is used. "Headspace bleed" labeled "ON" refers to the use of a stream of nitrogen gas directed through the headspace of the reaction vessel to purge headspace gas from the reaction vessel by the embodiments of the present disclosure described herein (e.g., directing a stream of pressurized nitrogen gas through an entry port as shown in Figures 1A and 1B, through the headspace into the reaction vessel, and out of the reaction vessel through an exit port such as a vent port as shown in Figures 1A and 1B). After the synthesis of the sequence is completed, the sequence is cleaved from the solid phase and the crude purity of the resulting sequence is analyzed. The results are reported in Table 2 below. [Table 2]

[0211] 42 The results for the β-amyloid and liraglutide sequences also show that high purity results are obtained without washing when headspace flushing is used as described herein for the respective deprotection steps.

[0212] Examples 6 and 7 above demonstrate that embodiments of the present disclosure including the deprotection step described herein (e.g., the deprotection step of the second embodiment) can provide an improved process for solid-phase peptide synthesis that can eliminate one or more (e.g., all) washing steps (e.g., can eliminate post-deprotection and / or post-coupling washing steps). In some embodiments, the process may use a small amount of deprotection base (e.g., about 3-4 vol.% pyrrolidine) for Fmoc removal, and / or heating (e.g., microwave heating at 80-110° C.), and / or removal of base from the deprotection solution by high temperature and / or nitrogen purging (bleeding the headspace), so that the remaining base is low enough to eliminate the need for washing before adding the next amino acid. Examples 6 and 7 demonstrate significant structural stability even with longer and more challenging sequences such as liraglutide. Thus, significant savings in solvent and time can be achieved with the present process.

[0213] In the above, examples of embodiments have been disclosed. The present invention is not limited to such exemplary embodiments. In the above, descriptions of the order of steps or other actions are described for the purpose of providing examples, and not for the purpose of limiting the scope of the disclosure (e.g., steps or actions may be performed in a different order than described, and steps and actions may be omitted and / or added, where appropriate). The drawings are schematic representations and therefore are not necessarily drawn to scale. Unless otherwise noted, certain terms are used in a generic and descriptive sense and not for purposes of limitation.

[0214] Numerical values ​​provided throughout this disclosure may be approximate, and for each range specified in this disclosure, all values ​​within the range (including endpoints) and all subranges within the range are also disclosed. Those skilled in the art will also readily understand that in various implementations of the features of this disclosure, reasonably different design tolerances (e.g., for numerical value(s)), precision, and / or accuracy may be applicable and appropriate to achieve a desired result. Thus, those skilled in the art will readily understand the meaning, usage, etc. of "substantially," "about," "approximately," and other terms herein. As a non-limiting example, the term "about" may indicate that the numerical value may vary by ±25%, such as ±20%, such as ±15%, such as ±10%, such as ±5%, such as ±4%, such as ±3%, such as ±2%, such as ±1%, such as less than ±1%, such as ±0.5%, such as less than ±0.5%, and may include all values ​​and subranges therebetween for each of the above ranges.

[0215] As used herein, the phrase "and / or" includes any combination of one or more of the associated listed items, in some embodiments optionally in combination with other elements not specifically identified by the phrase "and / or" (e.g., it can mean conjunctive elements in some embodiments and disjunctive elements in other embodiments). As a non-limiting example, "A and / or B" can mean A without B in some embodiments, B without A in some embodiments, both A and B in some embodiments, etc.

[0216] As used herein, the phrase "at least one" in reference to a list of one or more elements may mean at least one element selected from any one or more of the elements in the list of elements, but does not necessarily include at least one of every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. In some embodiments, elements, whether related or unrelated to the specifically identified elements, may optionally be present other than the elements specifically identified in the list of elements referred to by the phrase "at least one". As a non-limiting example, "at least one of A and B", "at least one of A or B", and / or "at least one of A and / or B" may mean in some embodiments at least one, optionally including more than one A, and no B (and optionally including elements other than B), in some embodiments at least one, optionally including more than one B, and no A (and optionally including elements other than A), in some embodiments at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements), and so forth.

[0217] As used herein, the indefinite articles "a" and "an" mean at least one ("a" and "an" can refer to singular and / or plural elements).

Claims

1. 1. A process for deprotecting protected amino acids during solid phase peptide synthesis, comprising: heating the protected amino acid and the deprotecting agent within a lower interior of the reaction vessel during a deprotection reaction that removes the protecting group from the protected amino acid, wherein the heating step volatilizes the deprotecting agent into an upper interior of the reaction vessel; and directing a first inert gas into the upper interior of the reaction vessel through a first opening in the top of the reaction vessel and out of the upper interior of the reaction vessel through a second opening in the top of the reaction vessel during the heating step to remove volatilized deprotecting agent from the interior of the reaction vessel. A process involving:

2. continuously directing the first inert gas through an upper interior of the reaction vessel during the heating step to remove volatilized deprotecting agent from the interior of the reaction vessel; and / or 10. The process of claim 1, comprising intermittently directing the first inert gas through an upper interior of the reaction vessel during the heating step to remove volatilized deprotecting agent from the interior of the reaction vessel.

3. the directing step includes directing the first inert gas downwardly through the first opening; or the directing step includes directing the first inert gas through the first opening to a spray head located inside the reaction vessel, the spray head fluidly connecting the first opening with the interior of the reaction vessel; the reaction vessel including at least one exterior sidewall extending around an exterior interior space; the spray head is positioned within the outer interior space and includes at least one inner sidewall extending around the inner interior space and a plurality of holes extending at least partially around the inner interior space; the inner and outer interior spaces are in fluid communication with each other through the holes of the plurality of holes; 2. The process of claim 1, wherein the directing step includes directing the first inert gas into the inner interior space through the first opening and out of holes of the plurality of holes, optionally toward the at least one outer sidewall, into the outer interior space.

4. 10. The process of claim 1, wherein the first inert gas is a pressurized inert gas.

5. 5. The process of claim 4, comprising directing the first inert gas at a first pressure through an upper interior of the reaction vessel and directing a second inert gas at a second pressure lower than the first pressure of the first inert gas into a lower interior of the reaction vessel.

6. 6. The process of claim 5, wherein the first pressure of the first inert gas ranges from about 1 psi to about 25 psi, and the second pressure of the second inert gas is less than the first pressure.

7. 10. The process of claim 1, wherein the reaction vessel is a microwave transparent reaction vessel and the heating step comprises heating the protected amino acid and the deprotecting agent using microwave irradiation.

8. 10. The process of claim 1, wherein the deprotecting agent comprises pyrrolidine.

9. 10. The process of claim 1, wherein the deprotecting agent comprises piperidine.

10. 10. The process of claim 1, wherein the heating step is carried out at a temperature of about 70°C to about 120°C.

11. the reaction vessel including at least one exterior sidewall extending around an interior space; a portion of the volatilized deprotection agent condenses on the sidewall; 2. The process of claim 1, wherein the directing step comprises directing the first inert gas toward the sidewall to move condensed deprotection agent downward toward a lower interior of the reaction vessel.

12. deprotecting the first protected amino acid according to the process of any one of claims 1 to 11; coupling a second amino acid to the deprotected amino acid to form a peptide from the first and second amino acids; and repeating the deprotecting and coupling steps to form a peptide comprising the first, second, and subsequent amino acids. Process for solid phase peptide synthesis.

13. 1. A process for deprotecting protected amino acids during solid phase peptide synthesis, comprising: removing the protecting group of the protected amino acid with a deprotection composition comprising a deprotecting agent in an amount of about 5 vol % or less based on the total volume of the deprotection composition; the protected amino acid and the deprotection composition are present in a lower interior of a reaction vessel and are heated during the step of removing the protecting group; at least a portion of the deprotecting agent evaporates into the upper interior of the reaction vessel during the removing step; and directing an inert gas through the interior of the reaction vessel during the step of removing the protecting group to remove vaporized deprotecting agent from the upper interior of the reaction vessel. A process involving:

14. 14. The process of claim 13, wherein the deprotection composition comprises the deprotection agent in an amount of about 2 vol % to about 5 vol %, based on the total volume of the deprotection composition.

15. 14. The process of claim 13, wherein the deprotection composition comprises the deprotection agent in an amount of about 2 vol % to about 4.5 vol %, based on the total volume of the deprotection composition.

16. 14. The process of claim 13, wherein the deprotection composition comprises the deprotection agent in an amount of about 3 vol% to about 4.5 vol%, based on the total volume of the deprotection composition.

17. the directing step includes introducing the inert gas into an upper interior of the reaction vessel through a first opening located at the top of the reaction vessel, and releasing the inert gas and vaporized deprotection agent from the upper interior of the reaction vessel through a second opening located at the top of the reaction vessel; or the directing step includes the steps of introducing the inert gas into a lower interior of the reaction vessel through an opening located at a lower portion of the reaction vessel, and releasing the inert gas and vaporized deprotecting agent from an upper interior of the reaction vessel through an opening located at an upper portion of the reaction vessel; or 14. The process of claim 13, wherein the directing step comprises introducing the inert gas into both an upper interior of the reaction vessel through a first opening located at an upper portion of the reaction vessel and a lower interior of the reaction vessel through an opening located at a lower portion of the reaction vessel, and releasing the inert gas and vaporized deprotecting agent from the upper interior of the reaction vessel.

18. 14. The process of claim 13, wherein the protected amino acid and the deprotection composition are heated at a temperature of about 40°C to about 120°C.

19. 14. The process of claim 13, wherein the protected amino acid and the deprotection composition are heated at a temperature of about 50°C to about 120°C.

20. 14. The process of claim 13, wherein the protected amino acid and the deprotection composition are heated at a temperature of about 70°C to about 120°C.

21. The process of claim 13, wherein the protected amino acid and the deprotecting agent are heated using microwave irradiation.

22. 14. The process of claim 13, wherein the deprotecting agent comprises pyrrolidine.

23. 14. The process of claim 13, wherein the deprotecting agent comprises piperidine.

24. continuously directing the inert gas through the interior of the reaction vessel; and / or 14. The process of claim 13, comprising intermittently directing the inert gas through the interior of the reaction vessel.

25. deprotecting the first protected amino acid according to the process of any of claims 13 to 24 to provide a deprotected amino acid; coupling a second amino acid to the deprotected amino acid to form a peptide from the first and second amino acids; and A process for solid phase peptide synthesis comprising repeating said deprotecting and coupling steps to form a peptide comprising said first, second and subsequent pluralities of amino acids.

26. 26. The process of claim 25, wherein the process does not include a washing step after the deprotecting step and before the coupling step.

27. 26. The process of claim 25, further comprising, after the deprotecting step and before the combining step, washing the interior of the reaction vessel with a washing composition in an amount less than the total volume of the deprotecting composition.

28. 28. The process of claim 27, comprising, after the deprotecting step and before the combining step, washing the interior of the reaction vessel with a washing composition in an amount less than half the total volume of the deprotecting composition.

29. 28. The process of claim 27, comprising, after the deprotecting step and before the combining step, washing the interior of the reaction vessel with a washing composition in an amount less than ⅓ of the total volume of the deprotecting composition.

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