TECHNICAL FIELD
The present disclosure relates to a new system and method for manufacturing
5 peptides synthetically. More specifically, the present disclosure relates to a device that
uses resin reactors in series as a mechanism for coupling peptides together as part of Solid
Phase Peptide Synthesis.
10
BACKGROUND
Solid Phase Peptide Synthesis ("SPPS") is the method and system that is most
commonly used to synthesize polypeptides and amino acid sequences. SPPS involves
coupling an activated amino acid (which is usually the terminal or last amino acid in the
sequence) to a solid support. This solid support is usually is a polymeric resin bead that is
functionalized (such as with an NH2 group). The terminal amino acid (which generally
15 has its NH2 terminus protected via a F-moc, BOC or other protecting group) is reacted
with the resin such that the functionalized group on the resin reacts with and binds to the
activated COOH group of the terminal amino acid. In this manner, the terminal amino
acid is covalently attached to the resin.
Then, in the next step, the NH2 terminus of the terminal amino acid is de-
20 protected, thereby exposing its NH2 group for the next reaction. Accordingly, a new
amino acid is introduced. This new amino acid has its NH2 terminus protected via a
protecting group (such as an Fmoc, BOC or another protecting group). As such, when this
new amino acid is added, the activated ester from the new amino acid reacts with the
newly de-protected NH2 group of the terminal amino acid, thereby coupling these two
25 amino acids together. Once this new amino acid has been coupled, it likewise has a
protected NH2 group that may be subsequently de-protected and reacted with the next
amino acid. By doing this repetitive, iterative process over and over, the entire amino
acid sequence may be constructed. Once the entire sequence has been constructed, the
sequence may be uncoupled (cleaved) from the resin and de protected, thereby producing
30 the amino acid structure. (It should be noted that the side chains of the various amino
acids (R1, R2, etc.) that are added via this process may be orthogonally protected via
groups such as BOC, t-butyl or trityl, etc. to prevent such side chains from reacting during
the amino acid synthesis process. Also, one or more of the amino acids may have a "side
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chain" or other group as part of its structure that may also have to be protected. However,
those skilled in the art will appreciate how such side chains or other group may be
constructed, protected, and subsequently de-protected during the synthesis process.)
While this SPPS process is used commercially and is still the standard in peptide
5 synthesis, it has a drawback in that it is expensive and time consuming. Each amino acid
that is added must be de-protected and coupled, which is difficult and usually results in
large quantities of solvents being used. Making matters worse is that many of these
solvents are not environmentally friendly.
Accordingly, it would be an improvement to find a new way to use SPPS, that
10 would address one or more of these deficiencies, especially in the commercial
manufacturing of peptides. It would be a further advancement if such a system could be
more environmentally friendly and reduce manufacturing costs. In fact, the present
embodiments will specifically reduce the quantity of waste, and the quantity of solvent
and reagents that are used. Such a method and system is disclosed herein.
15
SUMMARY
A process and system for coupling an amino acid "X" activated ester to a
protected N-group (such as an NH2 terminus) of an amino acid that is attached to a SPPS
resin. Generally, the system will comprise a collection of reactors that are arranged in
20 series. In some embodiments, two or more reactors are arranged in series. In a preferred
embodiment, 3 or more reactors are arranged in series.
Each reactor contains a quantity of a protected N-group affixed to a peptide
synthesis resin. This protected N-group may be an NH2 group of an amino acid or may be
an NH2 group found on or covalently attached to the resin itself such as, but not limited to
25 Sieber amide or Rink Amide resins. Other types of resins, such as Wang resins or CTC
(chlorotrityl chloride) resins may also be used.
The first step in the process involves adding a first quantity of de-protecting
reagent to the first reactor and allowing this reagent to contact the protected N-group.
Then, this first quantity of de-protecting reagent is transferred from the first reactor to the
30 second reactor and a second quantity of de-protecting reagent to the first reactor. The
first quantity of de-protecting reagent is removed from the second reactor and the second
quantity of de-protecting reagent is transferred from the first reactor to the second reactor.
This second quantity of de-protecting reagent is then removed from the second reactor.
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The purpose of contacting both the first and second reactors with the first and
second quantity of de-protecting reagent is so that this de-protecting reagent will react
with the protected N-group and, either separately or collectively, will operate to deprotect
the protected N-group affixed to the peptide synthesis resin in both the first and
5 second reactors. Thus, by adding the first and second quantity of de-protected reagent, the
N-group in both the first and second reactors are unprotected and capable of being
coupled to another amino acid. The first quantity of wash is added to the first reactor then
transferred to the second reactor, then to waste. The wash cycle is repeated several times.
The washing with solvent may be used with green solvents or solvents that are more
10 environmentally friendly that is typically used with SPPS. Such green wash solvents
include acetonitrile (ACN), ethyl acetate, isopropyl actetate, 2-MetHF (2-
Methyltetrahydrofuran), and CPME (cyclopentyl methyl ether),or solvent mixtures such
as NBP/THF 2/1 v/v, which is exemplified herein. The chemistry reactions could also
occur in ACN, ACN/DMSO or n-butylpyrrolidinone, which are also green solvents.
15 Accordingly, a first quantity amino acid "X" and a first quantity of solvent is
added to the first reactor and then after a certain amount of time, is transferred out of the
first reactor and into the second reactor. It should be noted that the quantities of amino
acid "X" that are added to the reactors are actually "activated esters" of the amino acid X,
thereby facilitating the coupling reaction. However, for shorthand notation, it may be
20 referred to herein as simply adding the "quantity of amino acid X" to the reactor, but
those skilled in the art will appreciate that it is the activated ester. Alternatively, the
unactiavated amino acid may be added to the reactor and then an activate solution is
added to react and convert the amino acid into an activated ester. That also falls within
the meaning of adding an amino acid activated ester to a resin, as used herein.
25 A second quantity of amino acid "X" and a second quantity of solvent is added to
the first reactor. The first and second quantity of amino acid "X" activated ester, either
separately or collectively, couples the amino acid "X" to the de-protected N group in the
first reactor. (Amino acid "X" activated ester may be any amino acid, including
functionalized, derivatized or synthetic amino acids that are desired to be added to the
30 chain). (As used herein, sometimes it is referred to that the amino acid "X" is coupled,
however, those skilled in the art will appreciate that it is the activated ester that is most
often used for easiness of reaction).
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The first quantity of amino acid "X" and the first quantity of solvent is removed
from the second reactor and the second quantity of amino acid "X" and the second
quantity of solvent is transferred from the first reactor to the second reactor. Then the
second quantity of amino acid "X" and the second quantity of solvent are removed the
5 from the second reactor. The first and second quantity of amino acid "X", either
separately or collectively, couples the amino acid "X" to the de-protected N group in the
second reactor. The first quantity of solvent wash is added to the first reactor then
transferred to the second reactor, then to waste. The solvent wash cycle is repeated
several times. The second quantity of solvent wash may be in the first reactor
10 simultaneously with the first quantity of solvent wash in the second reactor, and so on.
Thus, after performing these steps the amino acid "X" will have been coupled to the deprotected
N -group-thereby adding amino acid "X" to the chain. Of course, as with other
SPPS systems, amino acid "X" contains a protected NH2 group, and thus, the process
above may be repeated (e.g., de-protecting the NH2 group and coupling a new amino acid
15 to it, in the manner outlined above). Thus, by repeating this process, the desired amino
acid sequence and/or peptide may be constructed. Once the synthesis is finished, the
constructed amino acid may be released (de-coupled) to the resin in both the first reactor
and the second reactor.
Although the above-recited method uses two reactors in series, each with a supply
20 of resin, other embodiments may be designed in which a third reactor, also containing a
quantity of resin, is put in series with the first two reactors. In this embodiment, the deprotection
step must also occur in the third reactor. So once first quantity of de-protecting
reagent is removed from the second reactor, it is added to the third reactor. This first
quantity of de-protected reagent is removed from the third reactor, and then the second
25 quantity of de-protected agent, once it has been removed from the second reactor, is
added to the third reactor. Likewise, a third quantity of de-protecting reagent will be
added to the first reactor, moved the second reactor and then moved to the third reactor.
The purpose of the first, second and third quantity of de-protecting reagent, either
separately or collectively, is to de-protect the protected N-group affixed to the peptide
30 synthesis resin in the third reactor. In like manner, the first quantity of amino acid "X"
and the first quantity of solvent to the third reactor will be added to the third reactor after
it was removed from the second reactor. Similarly, the second quantity of amino acid "X"
and the second quantity of solvent to the third reactor will be added to the third reactor
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after it was removed from the second reactor. A third quantity of amino acid "X" and a
third third quantity of solvent is sequentially cycled through the first, second and third
reactors. The first, second and third quantity of amino acid "X", either separately or
collectively, couples the amino acid "X" to the de-protected N group in the third reactor.
5 A washing step, as described above, is then performed. Thus, in this manner, the amino
acid sequence may be built in all three reactors iteratively (by repeating these or similar
steps for each amino acid) and then released from the resins in each of the three reactors.
Thus, the present embodiments provide various reactors positioned in series and
that the reagents will be added to the first reactor and then subsequently and sequentially
10 moved to the second reactor and then the third reactor, etc. By positioning such reactors
in series, each reactor can contain a quantity of resin that will be used in SPPS which will
be used to construct a peptide sequence. Yet, by positioning the reactors in this manner,
lesser amount of solvent (washing material) will be required. Likewise, a lesser amount of
coupling reagent may be required, thus resulting in less waste and a more efficient and
15 environmentally-friendly process.
BRIEF DESCRIPTION OF THE DRAWINGS
The features and advantages of the present disclosure will become more apparent
to those skilled in the art upon consideration of the following detailed description taken in
20 conjunction with the accompanying figures.
25
FIG. 1 is a schematic view of the system and method for coupling amino acid "X"
to a protected N -group attached to a peptide synthesis resin used herein;
FIG. 2 is a schematic view of the system and method for coupling amino acid "X"
to a protected N -group attached to a peptide synthesis resin used herein;
FIG. 3 is a schematic view of the system and method for coupling amino acid "X"
to a protected N -group attached to a peptide synthesis resin used herein;
FIG. 4 is a schematic view of the system and method for online LCMS used with
the system of Figure 3;
FIG. 5 is a perspective view of three reactors in series as used in the present
30 embodiments;
FIG. 6 shows a peptide that may be made using the present embodiments; and
FIG. 7 shows a peptide that may be made using the present embodiments.
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DETAILED DESCRIPTION
For the purposes of promoting an understanding of the principles of the present
disclosure, reference will now be made to the embodiments illustrated in the drawings,
and specific language will be used to describe the same. It will nevertheless be
5 understood that no limitation of the scope of the invention is thereby intended.
Referring now to Figure 1, a schematic view of a system 100 for coupling amino
acid "X" to a protected N-group that is attached to a peptide synthesis resin. The system
100 implements the process outlined herein and is a modified SPPS system designed to
10 produce peptides and/or amino acid sequences. The system 100 comprise at least two
reactors, which are depicted as first reactor 106 and second reactor 108. These reactors
106, 108 are arranged in series. More than two reactors may be used. In fact, in the
system 100 shown in Figure 1, a third reactor 112 is also arranged in series. More than
three reactors may also be used.
15 Each of the reactors comprises a quantity of resin 116. The resin includes a
protected N-group 120 (such as a protected NH2) group. In some embodiments, this
protection group used for the protected N-group is an Fmoc group. Those skilled in the art
of SPPS will appreciate the types of resins that may be used as resin 116, including a
Seiber and Rink amide resin. As part of the SPPS process, the protected N-group must be
20 "de-protected" so that it can react with an amino acid (and thus operate to construct the
peptide/amino acid sequence). Thus, a de-protecting process occurs. This de-protection
occurs by adding a first quantity of de-protecting reagent 126. (This first quantity of deprotecting
reagent 126 is represented graphically by an arrow). In some embodiments, the
de-protection reagent may be piperidine, but other materials/reagents may also be used.
25 The first quantity of de-protecting reagent 126 may be stirred and allowed to react
with the protected N-group 120 on the resin 116 in the first reactor 106 for a period of
time. (Those skilled in the art will appreciate how to determine the exact time allotments
needed herein). Then, the first quantity of de-protecting reagent 126 is removed from the
first reactor 106 and transferred to the second reactor 108 (as shown by arrow 128). This
30 first quantity of de-protecting reagent 126 may be stirred and allowed to react with the
protected N-group 120 on the resin 116 in the second reactor 108 for a period of time. At
the same time, a second quantity of de-protecting reagent 130 is added to the first reactor
106 and allowed to react in a similar manner.
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Once these reactions have completed (or in other words, the allotted time has
expired), the first quantity of de-protecting reagent 126 is removed from the second
reactor 108 and transferred to the third reactor 112 (as shown by arrow 136). Likewise,
the second quantity of de-protecting reagent 130 is removed from the first reactor 106 and
5 transferred to the second reactor 108 (as shown by arrow 140). A third quantity of deprotecting
reagent 134 is then added to the first reactor 106. At this time, reactions in the
first reactor 106, the second reactor 108 and the third reactor 112 are allowed to continue.
After these reactions in the first reactor 106, the second reactor 108 and the third
reactor 112 finish (or in other words, the allotted time has expired), the first quantity of
10 de-protecting reagent 126 is removed from the third reactor 112. The second quantity of
de-protecting reagent 130 is removed from the second reactor 108 and transferred to the
third reactor 112 (as shown by arrow 144). The third quantity of de-protecting reagent
134 may be removed from the first reactor 106 and added to the second reactor 108 (as
shown by arrow 146).
15 In some embodiments, this first quantity of de-protecting reagent 126 that was
removed from the third reactor 112 may be sent to an additional reactor (if the
embodiment includes an additional reactor). In other embodiments, this first quantity of
de-protecting reagent 126 is collected and sent to waste. This will also happen to the
second and third quantities of de-protection reagents 130, 134 once they have cycled
20 through the third reactor 112. In other embodiments, including the embodiment shown in
Figure 1, this first quantity of de-protecting reagent 126 (and subsequently the second and
third de-protection reagents 130, 134) may be returned to the first reactor 106 (as shown
by arrow 148) so that additional iterations of the de-protection may be run as desired.
(Note that if the de-protection reagents are returned to the first reactor, this process will
25 be more like a batch reaction and will likely be less efficient).
The third quantity of reagent 134 will be removed from the first reactor 106 and
transferred to the second reactor 108. This quantity of reagent 134 will then cycle through
the second reactor 108 and the third reactor112, in the manner outlined herein. (although
the arrow for the third quantity of reagent 134 being sent from the second reactor 108 to
30 the third reactor 112 is not shown).
Those skilled in the art will appreciate that pumping corrections may be made to
either the first, second or third quantities of de-protecting reagents 126, 130, 134, as
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needed, in order to keep the volume of reagent in each quantity (and in each reactor)
uniform or nearly uniform.
As will be readily apparent to those skilled in the art, the purpose of the deprotection
reagents 126, 130, 134 that is added to the first, second and third reactors 106,
5 108, 112 is, either separately or collectively, to operate to de-protect the protected Ngroup
120 affixed to the peptide synthesis resins 116. By thus de-protecting theN-group
in series using quantities of reagent cycled through in series in the manner outlined
herein, theN-group may be ready for a coupling reaction which will operate to couple the
N-group to another amino acid (as will be described in greater detail below).
10 If desired, once the unprotected N-group that is attached to the resin 116 may be
"washed" with a solvent such as DMF. Other solvents may also be used including NBP
(N-butylpyrrolidinone), NMP (N-Methyl-2-pyrrolidone), DMSO, acetates and ethers like
MeTHF (methyltetrahydrofuran), or solvent mixtures such as NBP/THF 2/1 v/v, which is
exemplified herein. Such washing may occur in the same manner as outlined above with
15 respect to the de-protection reagents 126, 130, 134. In other words, a first quantity of
washing solvent may be added to the first reactor 106 and then subsequently cycled
through the second and third reactors 108, 112. Likewise, a second and third quantity of
washing solvent may likewise be cycled through reactors 106, 108 and 112. This washing
step may be iterative so that there may be 5, 8 or 10 different washing cycles (either with
20 the same quantity of solvent or with new quantities of solvent being added to the first
reactor each time). (Although not described in detail herein, the washing step may be
important and may be performed after each deprotection and coupling step). Likewise,
pump correction may also be used to ensure that each quantity of washing solvent that is
added to the reactors is nearly uniform.
25 Referring now to Figure 2, the coupling of the amino acid will now be described
using the system 100. Each of the resins 116 has been de-protected as outlined above (and
optionally washed). Accordingly, the resins 116 are shown to include unprotected Ngroup
120a (rather than protected N-group 120 that was shown in Figure 1).
A first quantity of amino acid "X" 150 is added to the first reactor 106. This is
30 represented graphically in Figure 2 by an arrow. In the embodiment shown in Figure 2,
the first quantity of amino acid "X" 150 has been pre-mixed with a first quantity of
solvent 152 as well as a first quantity of other reagents 154. More specifically, the first
quantity of amino acid "X" 150 has been mixed with the other reagents 154 and the
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solvent 152 prior to its addition to the first reactor 106. In other embodiments, the first
solvent 152 and/or the other reagents 154 may also be added sequentially and/or
simultaneously to the first reactor 106 in addition to the first quantity of amino acid "X"
150. In some embodiments, the first quantity of solvent 152 may be DMF. In some
5 embodiments, the other reagents 154 be agents that operate to "activate" the amino acid
"X" 150 and/or the unprotected N-group 120a and/or facilitate the coupling reaction.
Thus, in some embodiments, the other reagents 154 may be DIC and oxyma.
Once the amino acid "X" 150 (and the first quantity of solvent 152 and the other
reagents 154) are added to the first reactor 106, the coupling reaction is allowed to
10 proceed. This reaction occurs between the unprotected N -group 120a and the amino acid
"X". After a period of time (such as, for example 30 minutes or some other set amount of
time that those skilled in the art will understand how to calculate/determine), the first
quantity of amino acid "X" 150 may be removed from the first reactor 106 (as shown by
arrow 160). In some embodiments, this first quantity of amino acid "X" 150 may be
15 transferred to the second reactor 108. The first quantity of solvent 152 and/or the other
reagents 154 may also be removed from the first reactor 106 and transferred to the second
reactor 108.
Once in the second reactor 108, the first quantity of amino acid "X" (as well as the
first solvent 152 and/or the other reagents 154) may operate to react with the unprotected
20 N-group 120a in the second reactor 108. Likewise, a second quantity of amino acid "X"
170 may be added to the first reactor 106. (Again, this second quantity of amino acid "X"
170 may be pre-mixed with other reagents 154 and/or solvent 152). After a period of time
(such as, for example 30 minutes), the first quantity of amino acid "X" 150 may be
removed from the second reactor 108 (as shown by arrow 164). This first quantity of
25 amino acid "X" 150 may be transferred from the second reactor 108 to the third reactor
112. If a first quantity of solvent 152 and/or the other reagents 154 were used, then they
will be also be removed from the second reactor 108 and transferred to the third reactor
112. The second quantity of amino acid "X" 170 may be removed from the first reactor
106 (as shown by arrow 174). This second quantity of amino acid "X" 170 may be
30 transferred from the first reactor 106 to the second reactor 108.
A third quantity of amino acid "X" 180 may be added to the first reactor 106.
(Again, this third quantity of amino acid "X" 180 may be pre-mixed with other reagents
154 and/or solvent 152 and may be from the same batch as the first quantity 150 and/or
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the second quantity 170.) The reaction is allowed to proceed such that the unprotected Ngroup
120a in the first, second and third reactors 106, 108, 112 reacts with and is coupled
to the amino acid "X". Once this reaction is completed or after some period of time, the
first quantity of amino acid "X" may be removed from the third reactor 112 and sent to
5 waste or recycled back to the first reactor 106 (as indicated by arrow 188). (Again, such
recycling makes this reaction more like a batch, and thus may be less desired. In fact, if a
batch mode is desired, it may be better to couple the reactors in parallel). In like manner,
the second quantity of amino acid "X" 170 may be transferred to the third reactor 112
(and allowed to react as shown by arrow 166) and the first quantity of amino acid "X"
10 180 may be transferred to the second reactor 108 and subsequently to the third reactor
112 (and allowed to react in each reactor). In this iterative manner, the reaction occurs "in
series" and the first, second and third quantity of amino acid "X" 150, 170, 180 are cycled
through the reactors 106, 108, 112. In this manner, the amino acid quantities 150 170 180,
either separately or collectively, operates to react with the unprotected N-groups 120a and
15 couples the amino acid "X" to the de-protected N groups 120a in the first, second and
third reactors 106, 108, 112.
In some embodiments, it may be advantageous to filter the solution before it is
transferred from one reactor to the next reactor (for example, transferred from the first
reactor 106 to the second reactor 108 or from the second reactor 108 to the third reactor
20 112). Those skilled in the art will appreciate how such filtering may occur.
It will be appreciated that using reactors in series, as outlined herein, can provide a
reduction in the amount of solvent used for the de-protection and/or the washing steps.
For example, if traditional batch processing is used, a 20% solution of piperidine in DMF
25 would be required for the de-protection reaction. Such a solution would be divided into
10 volumes and each batch reactor reacted 3 times with this solution. This would result in
about 30 L/kg used for each reactor. However, if the three reactors are used in series, as
taught herein, the 20% solution of piperidine in DMF would still be divided into 10
volumes based on the resin amount in each reactor and reacted 3 times (iterations)
30 through the series of three reactors. In order for reactors 2 and 3 to experience number of
equivalents piperidine greater than or equal to batch, a fourth charge of 10 L/kg may be
used. Here, the overall amount of piperidine solution would be 40/3 = 13.3 L/kg, which is
a 2.25 fold reduction.
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The higher the number of excess amino acids and the higher the amino acid cost,
the more advantageous it is to use reactors in series for the couplings. For example, the
Lys20 IV decoupling reagent may be about $20/gram in price; thus, reductions in the
amount of excess reagents can result in significant cost savings. However, the lower the
5 number of excess equivalents, the more advantageous it is to run coupling reactions in
parallel, because of the hold up of reaction solution in the resin, preventing a percentage
of the excess equivalents from transporting to the next reactor without dilution. If the
excess amino acid equivalents are about 2 or less, and if they are standard inexpensive
amino acids, then we have chosen to run the coupling reactions in parallel but the
10 deprotections and washes in series.
It should be noted that, in some embodiments, the larger the number of reactors in
series, the less overall solvent and reagent is needed per kilogram of product. This is
analogous to the flow chemistry principle that a larger number of CSTRs (continuous
stirred-tank reactors in series becomes closer to ideal plug flow. In some embodiments, it
15 is believed that three reactors in series may be optimal because of the trade-off between
waste reduction and equipment cost. In other words, embodiments may be designed in
which diminishing returns are hit with more than 3 to 5 reactors in series. To achieve the
same maximum residual reagent concentration at the end of washing, 3 reactors in series
may operate to cut solvent requirements in half versus single batch processing. Of course,
20 other embodiments may be designed in which more than three or more than five reactors
are used. Further embodiments may be designed in which two reactors are used in series.
In some embodiments, it may be desirable to measure all the reagent charges by
mass and to use a Delta V distributive control system (provided by the Emerson company
of St. Louis Missouri USA) (rather than a Windows based user interfaces.) This because
25 documentation is also done automatically in Delta V and the system can create executed
master batch records. Further, in the Delta V system, most of the operation is done by
remote access, which can be accomplished from anywhere in the world that has internet.
The DeltaV system can be set to send descriptive text messages to the cell phones of
operators, chemists, engineers, and analysts when important steps are happening in the
30 process or when anything needs attention, which is subsequently handled remotely in
most circumstances. One of the common problems with the best commercially available
laboratory research scale SPPS technology is that control systems crash, miss charges, or
error-out at some point during the peptide build. Reagent charges are missed because they
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are done via level sensor. In contrast, the system can be designed to do all reagent charges
by mass from weigh scales. Individual experiments often need to run for more than a
month, for example if the peptide has more than 30 amino acids. An amino acid
mischarge near the end of a one-month experiment means that the experiment must be re-
5 started, and the month (or other time required for synthesis) is wasted, along with the
wasted materials. That is much less likely to happen with DeltaV automation because it is
designed to be more reliable and robust, as it is an industry standard for GMP
manufacturing.
In further embodiments, the system may be designed to provide much more
10 process information and understanding than other commercially available synthesizers
because of its online analytical. Online LCMS (liquid chromatography/mass
spectroscopy) of the peptide on resin, quantifies reaction conversion and kinetics for all
deprotections and couplings in all of the parallel reactors simultaneously. Timing is
integrated with the chemical process because the same Delta V system that runs the
15 process also runs the online analytical. Accordingly, workers may not need to be in the
lab to take and analyze samples for forward processing decisions.
In order to get an online LCMS system, the following steps may be implemented:
1. Pull 1.0 mL slurry from the resin reactor
2. Immediately cleave with TFA in small reactor (time from pulling the sample to
20 start cleavage with TFA is about 2 minutes)
3. Dilute cleaved peptide solution in LC (liquid chromatography) diluent and mix
4. Transport solution across the lab and park on the LC injection loop (gas-bubblefree)
5. Switch the loop to inject the sample on the LC
25 6. Flush the spent resin beads from the cleavage reactor to waste
7. Clean sample valves and tubing with solvent.
However, it should be noted that in order to implement this online LCMS, the flow goes
vertically up through the sample valves so that they get completely filled gas bubble free.
30 Two three-way valves are used to switch the slurry sample away from the sample loop
and blow it into the cleavage and deprotection zone. A single solenoid airline tees to the
actuators of both valves so that they switch at exactly the same time, which may be
important for getting exactly 1 mL of slurry every sample. Then, the slurry passes all the
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way through the sample zone and then continues in the uphill direction beyond of the
sample valves. This may be important so that the sample valves are filled with
representative slurry density, even when the viscosity and slurry density of the reactor are
changing from one step to the next and the flow distance past the valves changes. The
5 slurry sample tube from the reactor is uphill all the way to the sample valves. This is
important so that later the tube clears when pumping back to the reactor in reverse
direction. It minimizes carryover and prevents solid clogging. Solvent is used to flush out
the sample valves after every time the dilution cart finishes a sample. Otherwise, they
eventually clog with solids. The solvent enters between the sample valves and the
10 peristaltic pump in the backwards direction away from the valves, then pushes in the
forward direction through the valves. Solvent slugs flush all the way into the cleavage
zone to clear resin solids each time. Beyond the valves, the slurry continues to flow in the
uphill direction. The flow in this uphill direction is long enough so that there is enough
margin to ensure a representative sample gets in the sample loop but that the slurry does
15 not move past the apex and begin to flow back downhill. If it does, then it will get to the
peristaltic pump which can cause carryover problems and also grind up the resin, creating
filterability problems in the reactor. The peristaltic pump is located after the downhill part
of the sample loop instead of upstream from the sample loop. When it is upstream from
the sample loop it creates carryover problems and grinds up the resin causing reactor
20 filterability problems. A valve (which may be custom) may be used, with an extra port
welded on the body, so that the dilution solvent enters directly on top of the ball and
pushes in the upward direction, which completely mixes the dilution solvent with the
cleavage solution and also disrupts the resin bed settled on top of the ball.
Referring now to Figures 1 and 2 collectively, the addition of the next amino acid
25 in the peptide sequence will now be described. This next amino acid in the sequence may
be designated as "Z", meaning that it can be any amino acid desired (whereas, as noted
above, amino acid "X" may also be any amino acid desired). The steps and processes
outlined above will operate to couple amino acid "X" to the resin. (Thus, amino acid "X"
becomes the first amino acid in the peptide sequence). Once this amino acid "X" has been
30 coupled and attached to the resins, the resins in the first, second and/or third reactors 106,
108, 112 may be washed with solvent. This solvent will generally be the same solvent
outlined above. Such washing may occur in the sequential (e.g., in series) manner
outlined herein. Thus, for example, a first additional quantity of solvent may be added to
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the first reactor 106, stirred in the first reactor 106, and then transferred to the second
reactor 108 (and used to wash the resin in the second reactor 108, and then transferred to
the third reactor 112. Likewise, once the first additional quantity of solvent is removed
from the first reactor 106, a second additional quantity of solvent may be added to the
5 first reactor 106 (and cycled through the other reactors). (A third quantity of additional
solvent may also be likewise cycled through the system). In other words, the washing
step may proceed, in the sequence and manner outlined above.
Once the reactors 106, 108, 112 have been washed with solvent, a first additional
quantity of de-protecting reagent may be added to the first reactor 106 and then (after a
10 certain period of time), transferred out of the first reactor 106 and added to the second
reactor 108. This first additional quantity of de-protecting reagent will then (after a
certain period of time) also be removed from the second reactor 108. A second additional
quantity of de-protecting reagent is added to the first reactor 106, reacted for a period of
time, and then removed from the first reactor 106 and transferred to the second reactor
15 108, reacted therein and then removed from the second reactor 108 (and then added to the
third reactor 112 in the manner outlined herein). This first and second additional quantity
of de-protecting reagent (and third additional quantity of de-protected reagent), either
separately or collectively, operates to de-protect the protected N-group of amino acid "X"
in both the first and second reactors.
20 After this reaction with de-protecting reagent, the amino acid "X" (which is
25
affixed to the resin) is ready to be coupled to the next amino acid "Z" in the desired
sequence. Thus, in the manner outlined herein, the following steps will occur:
adding a first quantity amino acid "Z" to the first reactor 106;
transferring the first quantity of amino acid "Z" to the second reactor 108;
adding a second quantity of amino acid "Z" to the first reactor 106, wherein the
first and second quantity of amino acid "Z", either separately or collectively, couples the
amino acid "Z" to the de-protected N group of amino acid "X" in the first reactor 106;
removing the first quantity of amino acid "Z" from the second reactor 108; and
transferring the second quantity of amino acid "Z" from the first reactor 106 to the
30 second reactor 108; and
removing the second quantity of amino acid "Z" from the second reactor 108,
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wherein the first and second quantity of amino acid "Z", either separately or
collectively, couples the amino acid "Z" to the de-protected N group of amino acid "X" in
the second reactor 108.
Those skilled in the art will appreciate that the amino acid "Z" may also be
5 reacted with the amino acid "X" in the third reactor 112 in a like manner using these
series of reactors and reagents, adding each amino acid sequentially. Thus, in this
manner, the amino acid sequence is built. The process is then repeated, iteratively, to add
the next amino acid, and the next, etc., as in known is SPPS synthesis.
Referring now to Figure 3, a schematic view is shown of another embodiment of
10 a system 300 for coupling amino acid "X" to a protected N-group that is attached to a
peptide synthesis resin. Specifically, the system 300 includes multiple storage tanks 301
that are designed to house quantities of amino acids. Specifically, each specific amino
acid that will be added to the peptide chain may have its own separate storage tank 301.
Further, each tank 301 may have its own pump 303 which is designed to pump the amino
15 acid (which may be dissolved in a solution) out of the storage tank 301 and into an
activation reactor 307. Specifically, the pump 303 will pump the solution of the amino
acid out of the tank 301 through the line 309, into line 311, and into the reactor 307.
Those skilled in the art will appreciate how to connect this tubing/piping and the pump
303 to accomplish this this transfer of the amino acid solution from the individual tanks
20 301 to the reactor 307. Only 3 different amino acid tanks 301 are shown in Figure 3.
More may be used as desired.
Optionally, one or more flow sensors 313 may be attached to the line 309 and/or
the pumps 303 to sense the flow of the amino acid through the line (and into the reactor
307) so that the quantity, flow rate, flow timing, etc. may be adjusted, as desired. Further,
25 valves 317 may be used to divert the flow of the amino acid back to the feed vessel 301
for priming the pump. Those skilled in the art will appreciate how to use the valves and/or
the inerting vent system 327, which may also include a vent 329 as well as overflow
vessels for safety, as desired.
Similarly, the system 300 includes multiple storage tanks 353 that are designed to
30 house quantities of other reagents, such as DIC, oxyma, etc., and other wash solvents.
Further, each tank 353 may have its own pump 355 which is designed to pump the liquid
out of the storage tank 353 and into an activation reactor 307 or any of the filter reactors
306, 308, 312. Those skilled in the art will appreciate how to connect this tubing/piping
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and the pumps 355 to accomplish this this transfer. Only 3 different feed tanks 353 are
shown in Figure 3. More may be used as desired.
Optionally, one or more flow sensors 359 may be attached to the lines from
pumps 355 to sense the flow through the line so that the quantity, flow rate, flow timing,
5 etc. may be adjusted, as desired. Further, valves 357 may be used to divert the flow of the
back to the feed vessel353 for priming the pump. Those skilled in the art will appreciate
how to use the valves and/or the inerting system 363, which may also include a vent
bubbler 361 as well as overflow vessels for safety, as desired.
As noted above, the system 300 includes an activation reactor 307. The activation
10 reactor 307 is generally positioned upstream of the first reactor 306, the second reactor
308 and the third reactor 312. The activation reactor 307 may optionally include an
agitator 333 that is designed to mix the contents (solution) within the activation reactor
307. Also, a temperature probe 335 may also be added to the activation reactor 307. A
circulator 341 associated with the jacket 345 may also (optionally) be included.
15 The activation reactor 307 may be designed such that it "activates" the amino acid
solution by pre-mixing the amino acid solution with DIC and oxyma. Those skilled in the
art will appreciate how such addition of DIC and oxyma (and/or a solvent and some other
activating agent and base) may be added to the reactor 307.
The system 300 also includes a de-protection solution vessel371 which houses the
20 de-protecting reagent. (In the embodiment shown in Figure 3, the de-protecting reagent is
piperdine, although other materials may be used). One or more valves 369 and a pump
365 may be designed to pump the de-protecting reagent (via lines 367) to the first reactor
306 (via entry line 379). A relief valve 381 may optionally be added to the line 367.
Further, a pressure sensor 385 may optionally be used within the vessel371. Additional
25 valves 383 as part of a venting system for the vessel371 may optionally be used, as
desired.
A storage vessel371a used for solvent (which in this case may be DMF or some
other solvent) may also be used as part of the system 300. The storage vessel 371a may
include (optionally) a pressure sensor 373 to measure the pressure of the solvent. One or
30 more valves 375 and a pump 377 may be used to deliver the solvent vessel371a. The
solvent is delivered to the activation reactor 307, the first reactor 306, the second reactor
308 or the third reactor 312 via lines 381. A flow sensor 383 may be used (optionally) to
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measure this flow through the line 381. Additional venting valves 386 as desired may
optionally be used.

CLAIMS
1. A process for coupling amino acid "X" to a protected N-group attached to a
5 peptide synthesis resin comprising:
10
15
20
25
obtaining a first reactor and a second reactor, the first reactor and the second
reactor each containing a quantity of a protected N-group attached to a peptide synthesis
resin;
adding a first quantity of de-protecting reagent to the first reactor;
removing the first quantity of de-protecting reagent from the first reactor
adding the first quantity of de-protecting reagent to the second reactor;
adding a second quantity of de-protecting reagent to the first reactor;
removing the first quantity of de-protecting reagent from the second reactor;
removing the second quantity of de-protecting reagent from the first reactor;
adding the second quantity of de-protecting reagent to the second reactor;
removing the second quantity of de-protecting reagent from the second reactor;
washing the peptide synthesis resin in both the first and second reactors with a
solvent;
reactor;
reactor
reactor;
adding a first quantity amino acid "X" activated ester to the first reactor;
removing the first quantity amino acid "X" activated ester from the first reactor;
adding the first quantity of amino acid "X" activated ester to the second reactor,
adding a second quantity of amino acid "X" activated ester to the first reactor;
removing the first quantity of amino acid "X" activated ester from the second
removing the second quantity of amino acid "X" activated ester from the first
adding the second quantity of amino acid "X" activated ester to the second
removing the second quantity of amino acid "X" activated ester from the second
30 reactor; and
washing the peptide synthesis resin in both the first and second reactors with a
solvent.
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2. The process of claim 1, wherein the amino acid "X", that is found in the first and
second quantity of amino acid "X" activated ester, itself has a protected N-group.
3. The process of any of the preceding claims, further comprising:
adding the first quantity of de-protecting reagent to a third reactor, wherein this
adding occurs after the first quantity of de-protecting reagent is removed from the second
reactor, wherein the third reactor contains a quantity of a protected N-group attached to a
peptide synthesis resin; and
adding the second quantity of de-protecting reagent to the third reactor, wherein
10 this adding occurs after the second quantity of de-protecting reagent is removed from the
second reactor.
4. The process of claim 3, further comprising:
adding the first quantity of amino acid "X" activated ester to the third reactor,
15 wherein this adding occurs after the first quantity of amino acid "X" activated ester is
removed from the second reactor; and
20
adding the second quantity of amino acid "X" activated ester to the third reactor,
wherein this adding occurs after the second quantity of amino acid "X" activated ester is
removed from the second reactor.
5. The process of claim 4, further comprising:
adding a third quantity of de-protecting reagent to the first reactor, wherein this
third quantity of de-protecting reagent is added to the first reactor after the second
quantity of de-protecting reagent has been removed from the first reactor; and
25 transferring the third quantity of de-protecting reagent from the first reactor to the
30
second reactor, wherein this transferring occurs after the second quantity of de-protecting
reagent is removed from the second reactor; and
transferring the third quantity of de-protecting reagent from the second reactor to
the third reactor; and
removing the third quantity of de-protecting reagent from the third reactor.
6. The process of claim 5, further comprising:
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adding a third quantity amino acid "X" activated ester to the first reactor, wherein
this adding occurs after the second quantity of amino acid "X" activated ester is removed
from the first reactor;
transferring the third quantity of amino acid "X" activated ester to the second
5 reactor, wherein this transferring occurs after the second quantity of amino acid "X"
activated ester is removed from the second reactor;
transferring the third quantity of amino acid "X" activated ester from the second
reactor to the third reactor; and
removing the third quantity of amino acid "X" activated ester from the third
10 reactor.
7. The process of claim 6, further comprising:
removing the first quantity of amino acid "X" activated ester from the third
reactor; and
15 adding the first quantity of amino acid "X" activated ester back to first reactor.
20
8. The process of claim 7, further comprising:
removing the first quantity of de-protecting reagent from the third reactor; and
adding the first quantity of de-protecting reagent back to the first reactor.
9. The process of any of the preceding claims, further comprising swelling the first
and second reactors.
10. The process of any of the preceding claims, wherein the first quantity of amino
25 acid "X" activated ester and the second quantity of amino acid "X" activated ester are
pre-mixed with a solvent.
30
11. The process of any of the preceding claims, wherein the washing the first and
second reactors with a solvent occurs by adding solvent to the reactors.
12. The process of claim 2, wherein after the second quantity of amino acid "X"
activated ester has been removed from the first reactor, the method further comprising:
adding a first additional quantity of de-protecting reagent to the first reactor;
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transferring the first additional quantity of de-protecting reagent from the first
reactor to the second reactor;
adding a second additional quantity of de-protecting reagent to the first reactor;
removing the first additional quantity of de-protecting reagent from the second
5 reactor;
10
transferring the second additional quantity of de-protecting reagent from the first
reactor to the second reactor;
removing the second additional quantity of de-protecting reagent from the second
reactor.
13. The process of claim 12 wherein after the second additional quantity of deprotecting
reagent is removed from the first reactor, further comprising:
adding a first quantity amino acid "Z" activated ester to the first reactor;
transferring the first quantity of amino acid "Z" activated ester to the second
15 reactor;
reactor;
adding a second quantity of amino acid "Z" activated ester to the first reactor;
removing the first quantity of amino acid "Z" activated ester from the second
transferring the second quantity of amino acid "Z" activated ester from the first
20 reactor to the second reactor; and
25
removing the second quantity of amino acid "Z" activated ester from the second
reactor.
14. The process of claim 13, further comprising:
adding the first additional quantity of de-protecting reagent to a third reactor,
wherein this adding occurs after the first additional quantity of de-protecting reagent is
removed from the second reactor, wherein the third reactor contains a quantity of a
protected N-group of amino acid "X" activated ester; and
adding the second additional quantity of de-protecting reagent to the third reactor,
30 wherein this adding occurs after the second quantity of de-protecting reagent is removed
from the second reactor.
15. The process of claim 14, further comprising:
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adding the first quantity of amino acid "Z" activated ester to the third reactor,
wherein this adding occurs after the first quantity of amino acid "Z" is removed from the
second reactor; and
adding the second quantity of amino acid "Z" activated ester to the third reactor,
5 wherein this adding occurs after the second quantity of amino acid "Z" activated ester is
removed from the second reactor.
16. The process of any of the preceding claims, wherein the peptide synthesis resin in
the first and second reactors are Seiber or Rink resins.
10
17. The process of any of the preceding claims, wherein the peptide synthesis resin in
the first and second reactors are Wang resins or CTC resins.
18. The process of any of the preceding claims, wherein the amount of amino acid X
15 that is added is between 1.1 and 1.6 equivalents.
20
25
30
19. A process for coupling amino acid "X" to a protected N-group that is attached to a
peptide synthesis resin found in a first reactor and a second reactor, comprising:
reactor;
adding a first quantity of de-protecting reagent to the first reactor;
removing the first quantity of de-protecting reagent from the first reactor
adding the first quantity of de-protecting reagent to the second reactor;
adding a second quantity of de-protecting reagent to the first reactor;
removing the first quantity of de-protecting reagent from the second reactor;
removing the second quantity of de-protecting reagent from the first reactor
adding the second quantity of de-protecting reagent to the second reactor;
removing the second quantity of de-protecting reagent from the second reactor;
adding a first quantity amino acid "X" activated ester to the first reactor;
removing the first quantity amino acid "X" activated ester from the first reactor;
adding the first quantity of amino acid "X" activated ester to the second reactor;
adding a second quantity of amino acid "X" activated ester to the first reactor;
removing the first quantity of amino acid "X" activated ester from the second
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removing the second quantity of amino acid "X" activated ester from the first
reactor;
adding the second quantity of amino acid "X" activated ester to the second
reactor; and
5 removing the second quantity of amino acid "X" activated ester from the second
10
reactor.
20. The process of claim 19, wherein the amino acid "X", that is found in the first and
second quantity of amino acid "X" activated ester, itself has a protected N-group.
21. The process of either claim 18 or 19, further comprising:
adding the first quantity of de-protecting reagent to a third reactor, wherein this
adding occurs after the first quantity of de-protecting reagent is removed from the second
reactor, wherein the third reactor contains a quantity of a protected N-group attached to a
15 peptide synthesis resin;
removing the first quantity of de-protecting reagent from the third reactor;
adding the second quantity of de-protecting reagent to the third reactor, wherein
this adding occurs after the second quantity of de-protecting reagent is removed from the
second reactor;
20 adding a third quantity of de-protecting reagent to the first reactor, wherein this
third quantity of de-protecting reagent is added to the first reactor after the second
quantity of de-protecting reagent has been removed from the first reactor; and
transferring the third quantity of de-protecting reagent from the first reactor to the
second reactor, wherein this transferring occurs after the second quantity of de-protecting
25 reagent is removed from the second reactor;
transferring the third quantity of de-protecting reagent from the second reactor to
the third reactor;
adding the first quantity of amino acid "X" activated ester to the third reactor,
wherein this adding occurs after the first quantity of amino acid "X" activated ester is
30 removed from the second reactor;
removing the first quantity of amino acid "X" activated ester from the third
reactor;
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adding the second quantity of amino acid "X" activated ester to the third reactor,
wherein this adding occurs after the first quantity of amino acid "X" activated ester is
removed from the third reactor; and
removing the second quantity of amino acid "X" activated ester from the third
5 reactor.
22. The process of claim 21, further comprising:
adding a third quantity amino acid "X" activated ester to the first reactor, wherein
this adding occurs after the second quantity of amino acid "X" activated ester is removed
10 from the first reactor;
transferring the third quantity of amino acid "X" activated ester to the second
reactor, wherein this transferring occurs after the second quantity of amino acid "X"
activated ester is removed from the second reactor;
transferring the third quantity of amino acid "X" activated ester and from the
15 second reactor to the third reactor;
removing the third quantity of amino acid "X" activated ester from the third
reactor;
adding the first quantity of amino acid "X" activated ester back to first reactor,
wherein this adding of the first quantity of amino acid "X" activated ester occurs after the
20 first quantity of amino acid "X" activated ester is removed from the third reactor.
23. A process for coupling amino acid "X" to a de-protected N-group attached to a
peptide synthesis resin comprising:
obtaining a first reactor and a second reactor, the first reactor and the second
25 reactor each containing a quantity of a de-protected N-group attached to a peptide
30
synthesis resin;
reactor;
adding a first quantity amino acid "X" activated ester to the first reactor;
removing the first quantity amino acid "X" activated ester from the first reactor;
adding the first quantity of amino acid "X" activated ester to the second reactor,
adding a second quantity of amino acid "X" activated ester to the first reactor;
removing the first quantity of amino acid "X" activated ester from the second
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removing the second quantity of amino acid "X" activated ester from the first
reactor;
adding the second quantity of amino acid "X" activated ester to the second
reactor; and
5 removing the second quantity of amino acid "X" activated ester from the second
10
reactor.
24. The process of claim 23, further comprising washing the first reactor and a second
reactor with solvent.
25. The process of claim 22 or 23, further comprising:
adding the first quantity of amino acid "X" activated ester to a third reactor,
wherein this adding occurs after the first quantity of amino acid "X" is removed from the
second reactor, wherein the third reactor contains a quantity of a protected N-group
15 attached to a peptide synthesis resin;
20
removing the first quantity of amino acid "X" activated ester from the third
reacto;r and
adding the second quantity of amino acid "X" to the third reactor.
26. The process of claim 25, further comprising:
adding a third quantity of amino acid "X" to the first reactor, wherein this third
quantity of amino acid "X" is added to the first reactor after the second quantity of amino
acid "X" has been removed from the first reactor; and
transferring the third quantity of amino acid "X" from the first reactor to the
25 second reactor, wherein this transferring occurs after the second quantity of amino acid
"X" is removed from the second reactor.
30
27. The process of claim 26, further comprising:
removing the second quantity of amino acid "X" from the third reactor;
transferring the third quantity of amino acid "X" from the second reactor to the
third reactor;
removing the third quantity of amino acid "X" from the third reactor; and
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10
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adding the first quantity of amino acid "X" back to the first reactor, wherein the
first quantity of amino acid "X" is added back to the first reactor after the first quantity of
amino acid "X" de-protecting reagent has been removed from the third reactor.
28. A process for de-protecting a protected N-group attached to a peptide synthesis
resin comprising:
obtaining a first reactor and a second reactor, the first reactor and the second
reactor each containing a quantity of a protected N-group attached to a peptide synthesis
resin;
adding a first quantity of de-protecting reagent to the first reactor;
removing the first quantity of de-protecting reagent from the first reactor
adding the first quantity of de-protecting reagent to the second reactor;
adding a second quantity of de-protecting reagent to the first reactor;
removing the first quantity of de-protecting reagent from the second reactor;
removing the second quantity of de-protecting reagent from the first reactor;
adding the second quantity of de-protecting reagent to the second reactor; and
removing the second quantity of de-protecting reagent from the second reactor.
29. The process of claim 28, further comprising washing the first reactor and a second
20 reactor with solvent.
30. The process of claim 28 or 29, further comprising:
adding the first quantity of de-protecting reagent to a third reactor, wherein this
adding occurs after the first quantity of de-protecting reagent is removed from the second
25 reactor, wherein the third reactor contains a quantity of a protected N-group attached to a
peptide synthesis resin;
removing the first quantity of de-protecting reagent from the third reactor; and
adding the second quantity of de-protecting reagent to the third reactor, wherein
this adding occurs after the second quantity of de-protecting reagent is removed from the
30 second reactor.
31. The process of claim 30, further comprising:
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adding a third quantity of de-protecting reagent to the first reactor, wherein this
third quantity of de-protecting reagent is added to the first reactor after the second
quantity of de-protecting reagent has been removed from the first reactor; and
transferring the third quantity of de-protecting reagent from the first reactor to the
5 second reactor, wherein this transferring occurs after the second quantity of de-protecting
reagent is removed from the second reactor.
10
32. The process of claim 31, further comprising:
removing the second quantity of de-protecting reagent from the third reactor;
transferring the third quantity of de-protecting reagent from the second reactor to
the third reactor;
removing the third quantity of de-protecting reagent from the third reactor; and
adding the first quantity of de-protecting reagent back to the first reactor, wherein
the first quantity of de-protecting reagent is added back to the first reactor after the first
15 quantity of de-protecting reagent has been removed from the third reactor.
33. A system for de-protecting a protected N-group attached to a peptide synthesis
resin comprising a first reactor and a second reactor, the first reactor and the second
reactor each containing a quantity of a protected N-group attached to a peptide synthesis
20 resin.
34. A system for coupling amino acid "X" to a de-protected N-group attached to a
peptide synthesis resin comprising a first reactor and a second reactor, the first reactor
and the second reactor each containing a quantity of a protected N-group attached to a
25 peptide synthesis resin.
35. A system for coupling amino acid "X" to a de-protected N-group attached to a
peptide synthesis resin comprising a first reactor and an LCMS device, wherein the
system automatically samples slurry from the reactor,and cleaves the peptide from resin
30 and deprotects the functional groups.