Note: Descriptions are shown in the official language in which they were submitted.
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REACTOR SYSTEM AND METHODS FOR USING THEREOF
BACKGROUND
[0001] Reactors refer to any manufactured device or system in which chemical
or biochemical
processes can be carried out, including the manufacturing of therapeutic, such
as biological or
small molecule, products. There is a need for reactors that enable continuous
manufacturing
process for Active Pharmaceutical Ingredients (API) and/or biopharmaceuticals
through increased
flexibility and lower operational costs.
SUMMARY
[0002] The present disclosure provides methods and systems for the continuous
production and
processing of compounds, including biopharmaceutical compounds.
Biopharmaceutical
compounds may include therapeutic and/or prophylactic substances that require
sequential
processing steps, including synthesis, isolation, purification and packaging.
In some aspects, the
biopharmaceutical compounds include nucleic acid and biological
macromolecules.
[0003] In an aspect, the present disclosure provides a system for producing a
compound,
comprising: a plurality of chambers, wherein each chamber of the plurality of
chambers is
configured to contain at least one reagent for producing the compound; at
least one robotic arm
configured to load each chamber with the at least one reagent; and at least
one computer processor
operatively coupled to the plurality of chambers and the at least one robotic
arm, wherein the at
least one robotic arm is programmable to: (a) fill a first chamber with the at
least one reagent; (b)
fill a second chamber after a time interval X with the at least one reagent;
and (c) repeat step (b)
until at least a portion of the plurality of chambers is filled with the at
least one reagent, wherein
the compound is produced and removed after a time interval Y from each of the
portion of plurality
of chambers in step (c), and wherein the system is programmable in at least a
portion of the plurality
of chambers to re-load, produce and remove the compound as in steps (a)-(c),
thereby for a time
period of at least 2Y continuously producing the compound within at least a
portion of the plurality
of chambers.
[0004] In some embodiments, the plurality of chambers is housed in a
cartridge. In some
embodiments, the plurality of chambers is disposed on a conveyor. In some
embodiments, a
number of the plurality of chambers is at least the time interval Y divided by
the time interval X.
In some embodiments, the compound is continuously produced for a time period
of at least 3Y
within at least a portion of the plurality of chambers. In some embodiments,
the compound is
continuously produced for a time period of at least 4Y within at least a
portion of the plurality of
chambers. In some embodiments, the compound is continuously produced for a
time period of at
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least 5Y within at least a portion of the plurality of chambers. In some
embodiments, the compound
is continuously produced for a time period of at least 6Y within at least a
portion of the plurality
of chambers. In some embodiments, the compound is continuously produced for a
time period of
at least 7Y within at least a portion of the plurality of chambers. In some
embodiments, the
compound is continuously produced for a time period of at least 8Y within at
least a portion of the
plurality of chambers. In some embodiments, the compound is continuously
produced for a time
period of at least 9Y within at least a portion of the plurality of chambers.
In some embodiments,
the compound is continuously produced for a time period of at least 10Y within
at least a portion
of the plurality of chambers. In some embodiments, the compound is
continuously produced for a
time period of at least 20Y within at least a portion of the plurality of
chambers. In some
embodiments, the compound is continuously produced for a time period of at
least 30Y within at
least a portion of the plurality of chambers. In some embodiments, the
compound is continuously
produced for a time period of at least 40Y within at least a portion of the
plurality of chambers. In
some embodiments, the compound is continuously produced for a time period of
at least 50Y
within at least a portion of the plurality of chambers. In some embodiments,
the compound is
continuously produced for a time period of at least 60Y within at least a
portion of the plurality of
chambers. In some embodiments, the compound is continuously produced for a
time period of at
least 70Y within at least a portion of the plurality of chambers. In some
embodiments, the
compound is continuously produced for a time period of at least 80Y within at
least a portion of
the plurality of chambers. In some embodiments, the compound is continuously
produced for a
time period of at least 90Y within at least a portion of the plurality of
chambers. In some
embodiments, the compound is continuously produced for a time period of at
least 100Y within at
least a portion of the plurality of chambers. In some embodiments, the
compound is continuously
produced for a time period of at least 200Y within at least a portion of the
plurality of chambers.
In some embodiments, the compound is continuously produced for a time period
of at least 300Y
within at least a portion of the plurality of chambers. In some embodiments,
the compound is
continuously produced for a time period of at least 400Y within at least a
portion of the plurality
of chambers. In some embodiments, the compound is continuously produced for a
time period of
at least 500Y within at least a portion of the plurality of chambers. In some
embodiments, the
compound is continuously produced for a time period of at least more than 5Y
within at least a
portion of the plurality of chambers. In some embodiments, the compound is
continuously
produced for a time period of at least more than 6Y within at least a portion
of the plurality of
chambers. In some embodiments, the compound is continuously produced for a
time period of at
least more than 7Y within at least a portion of the plurality of chambers. In
some embodiments,
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the compound is continuously produced for a time period of at least more than
8Y within at least
a portion of the plurality of chambers. In some embodiments, the compound is
continuously
produced for a time period of at least more than 9Y within at least a portion
of the plurality of
chambers. In some embodiments, the compound is continuously produced for a
time period of at
least more than 10Y within at least a portion of the plurality of chambers. In
some embodiments,
the compound is continuously produced for a time period of at least more than
20Y within at least
a portion of the plurality of chambers. In some embodiments, the compound is
continuously
produced for a time period of at least more than 30Y within at least a portion
of the plurality of
chambers. In some embodiments, the compound is continuously produced for a
time period of at
least more than 40Y within at least a portion of the plurality of chambers. In
some embodiments,
the compound is continuously produced for a time period of at least more than
50Y within at least
a portion of the plurality of chambers. In some embodiments, the compound is
continuously
produced for a time period of at least more than 60Y within at least a portion
of the plurality of
chambers. In some embodiments, the compound is continuously produced for a
time period of at
least more than 70Y within at least a portion of the plurality of chambers. In
some embodiments,
the compound is continuously produced for a time period of at least more than
80Y within at least
a portion of the plurality of chambers. In some embodiments, the compound is
continuously
produced for a time period of at least more than 90Y within at least a portion
of the plurality of
chambers. In some embodiments, the compound is continuously produced for a
time period of at
least more than 100Y within at least a portion of the plurality of chambers.
In some embodiments,
the compound is continuously produced for a time period of at least more than
200Y within at least
a portion of the plurality of chambers. In some embodiments, the compound is
continuously
produced for a time period of at least more than 300Y within at least a
portion of the plurality of
chambers. In some embodiments, the compound is continuously produced for a
time period of at
least more than 400Y within at least a portion of the plurality of chambers.
In some embodiments,
the compound is continuously produced for a time period of at least more than
500Y within at least
a portion of the plurality of chambers. In some embodiments, the cartridge
comprises at least about
2 chambers. In some embodiments, the cartridge comprises at least about 3
chambers. In some
embodiments, the cartridge comprises at least about 4 chambers. In some
embodiments, the
cartridge comprises at least about 5 chambers. In some embodiments, the
cartridge comprises at
least about 6 chambers. In some embodiments, the cartridge comprises at least
about 7 chambers.
In some embodiments, the cartridge comprises at least about 8 chambers. In
some embodiments,
the cartridge comprises at least about 9 chambers. In some embodiments, the
cartridge comprises
at least about 10 chambers. In some embodiments, the cartridge comprises at
least about 12
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chambers. In some embodiments, the cartridge comprises at least about 20
chambers. In some
embodiments, the cartridge comprises at least about 24 chambers. In some
embodiments, the
cartridge comprises at least about 30 chambers. In some embodiments, the
cartridge comprises at
least about 36 chambers. In some embodiments, the cartridge comprises at least
about 40 chambers.
In some embodiments, the cartridge comprises at least about 48 chambers. In
some embodiments,
the cartridge comprises at least about 50 chambers. In some embodiments, the
chamber is
removable from the cartridge. In some embodiments, the chamber is not
removable from the
cartridge. In some embodiments, the chamber is removable from the conveyor. In
some
embodiments, the chamber is not removable from the conveyor. In some
embodiments, the
chamber comprises a volume of about at least 0.1 ml to at least about 1000 mL.
In some
embodiments, the chamber comprises a volume of about at least 1 mL. In some
embodiments, the
chamber comprises a volume of about at least 5 mL. In some embodiments, the
chamber comprises
a volume of about at least 10 mL. In some embodiments, the chamber comprises a
volume of at
least about 10 mL. In some embodiments, the chamber is pie wedge shaped. In
some embodiments,
the chamber is regular or irregular polygon shaped. In some embodiments, the
chamber is regular
or irregular hexagon shaped. In some embodiments, the chamber is circular
shaped. In some
embodiments, the chamber is regular or elliptic shaped. In some embodiments,
the chamber
comprises at least one baffle. In some embodiments, the first chamber touches
at least one side
wall of the second chamber. In some embodiments, the first chamber does not
touch any side wall
of the second chamber. In some embodiments, when the last chamber of the
plurality of chambers
is filled with the at least one reagent, the compound is produced and removed
from the first
chamber of the plurality of chambers.
[0005] In some embodiments, the time interval Y is at least the time interval
X times the number
of the plurality of chambers. In some embodiments, the time interval X
comprises at least about 2
minutes. In some embodiments, the time interval X comprises at least about 3
minutes. In some
embodiments, the time interval X comprises at least about 4 minutes. In some
embodiments, the
time interval X comprises at least about 5 minutes. In some embodiments, the
time interval X
comprises at least about 10 minutes. In some embodiments, the time interval X
comprises at least
about 15 minutes. In some embodiments, the time interval X comprises at least
about 20 minutes.
In some embodiments, the time interval X comprises at least about 25 minutes.
In some
embodiments, the time interval X comprises at least about 30 minutes. In some
embodiments, the
time interval X comprises at least about 35 minutes. In some embodiments, the
time interval X
comprises at least about 40 minutes. In some embodiments, the time interval X
comprises at least
about 45 minutes. In some embodiments, the time interval X comprises at least
about 50 minutes.
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In some embodiments, the time interval X comprises at least about 55 minutes.
In some
embodiments, the time interval X comprises at least about 60 minutes. In some
embodiments, the
time interval Y comprises at least about 30 minutes. In some embodiments, the
time interval Y
comprises at least about 60 minutes. In some embodiments, the time interval Y
comprises at least
5 about 75 minutes. In some embodiments the time interval Y comprises at
least about 90 minutes.
In some embodiments, the time interval Y comprises at least about 120 minutes.
In some
embodiments, the time interval Y comprises at least about 180 minutes. In some
embodiments, the
time interval Y comprises at least about 210 minutes. In some embodiments, the
time interval Y
comprises at least about 240 minutes. In some embodiments, the time interval Y
comprises at least
about 270 minutes. In some embodiments, the time interval Y comprises at least
about 300 minutes.
In some embodiments, the time interval Y comprises at least about 330 minutes.
In some
embodiments, the time interval Y comprises at least about 360 minutes. In some
embodiments, the
time interval Y comprises at least about 420 minutes. In some embodiments, the
time interval Y
comprises at least about 480 minutes. In some embodiments, the time interval Y
comprises at least
about 720 minutes. In some embodiments, the time interval Y comprises at least
about 1,440
minutes. In some embodiments, the compound is removed by the at least one
robotic arm. In some
embodiments, the compound is removed by a pressurized system. In some
embodiments, the
pressurized system comprises a negative pressure. In some embodiments, the
pressurized system
comprises a pump configured to remove a medium comprising the compound. In
some
embodiments, the pump is in fluid communication with a chamber. In some
embodiments, the first
chamber is not in fluid communication with the second chamber. In some
embodiments, the
chamber is configured to conduct an in vitro transcription (IVT) reaction. In
some embodiments,
the compound is a RNA molecule. In some embodiments, a reagent comprises a DNA
template. In
some embodiments, the DNA template is circular. In some embodiments, the DNA
template is
linear. In some embodiments, the chamber is configured to conduct an in vitro
translation. In some
embodiments, the in vitro translation is cell dependent. In some embodiments,
the in vitro
translation is cell free. In some embodiments, the chamber is not designed to
culture a living cell.
[0006] In some embodiments, the chamber is configured to monitor and/or
regulate a pH value of
a medium comprising the at least one reagent. In some embodiments, the chamber
is configured to
.. monitor and /or regulate a temperature of a medium comprising the at least
one reagent or an
atmosphere within the chamber. In some embodiments, the chamber is configured
to monitor
and/or regulate a salt concentration of a medium comprising the at least one
reagent. In some
embodiments, the chamber is configured to monitor and/or regulate osmolarity
of a medium
comprising the at least one reagent. In some embodiments, the chamber is
configured to monitor
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and/or regulate conductivity of a medium comprising the at least one reagent.
In some
embodiments, the chamber is configured to monitor and/or regulate a turbidity
of the medium
comprising the at least one reagent. In some embodiments, the chamber is
configured to monitor
and/or regulate a volume of the medium comprising the at least one reagent. In
some embodiments,
the chamber is configured to monitor and/or regulate a humidity of the
atmosphere within the
chamber. In some embodiments, the chamber is configured to monitor and/or
regulate an 02
concentration or CO2 concentration of the atmosphere within the chamber. In
some embodiments,
the chamber is configured to monitor and/or regulate a concentration of a
molecule comprised in
the medium utilizing a spectroscopic device. In some embodiments, the
spectroscopic device
comprises an Infrared spectrometer, a Raman spectrometer, or a UV
spectrometer.
[0007] In some embodiments, the system is programmable to inspect the medium.
In some
embodiments, the chamber comprises an agitation device. In some embodiments,
the agitation
device comprises a baffle, a magnetic bar, an impeller, or a bead. In some
embodiments, the
cartridge is programmable to shake to mix the medium. In some embodiments, the
chamber
comprises a lid. In some embodiments, the lid is removable. In some
embodiments, the lid is not
removable. In some embodiments, the lid comprises at least one opening. In
some embodiments,
a chamber is configured to detect a contamination of the at least one reagent.
In some embodiments,
the system further comprises at least one intermediate storage vessel. In some
embodiments, the
intermediate storage vessel is configured to store the compound. In some
embodiments, the system
comprises a purification system. In some embodiments, the purification system
comprises an
affinity purification system. In some embodiments, the purification system
comprises an ion
exchange system. In some embodiments, the purification system comprises a
selective
precipitation system. In some embodiments, the purification system comprises a
chromatography
system. In some embodiments, the purification system comprises a tangential
flow filtration device
or a dead-end filtration device. In some embodiments, the intermediate storage
vessel is not in fluid
communication with the chamber. In some embodiments, the storage vessel is
configured to detect
a contamination of the compound.
[0008] In some embodiments, the system further comprises a modular unit. In
some embodiments,
the modular unit comprises a compartment. In some embodiments, the compartment
comprises at
least one cartridge. In some embodiments, the at least one cartridge is
stackable. In some
embodiments, the modular unit comprises at least one carousel. In some
embodiments, the at least
on carousel is stackable. In some embodiments, the modular unit comprises at
least one conveyor.
In some embodiments, the at least one conveyor is stackable. In some
embodiments, the modular
unit is movable. In some embodiments, the modular unit is stackable. In some
embodiments, the
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modular unit comprises a plurality of receiving containers. In some
embodiments, the receiving
container is configured to contain at least one reagent. In some embodiments,
the modular unit
comprises a docking station configured to receive a receiving container, a
cartridge, a carousel, or
a conveyor. In some embodiments the modular unit is mobile. In some
embodiments, the modular
.. unit is non-mobile. In some embodiments, the modular unit is configured to
agitate contents (e.g.,
medium comprise in a chamber) disposed therein. In some cases, the modular
unit is configured to
shake or oscillate, thereby mixing contents disposed therein. In some
embodiments, the modular
unit is configured to regulate one or more properties of a chamber, cartridge,
conveyor, or carousel
disposed therein. In some embodiments, the modular unit is configured to
regulate a temperature
of the cartridge or chamber. In some embodiments, the system further comprises
a second robotic
arm. In some embodiments, the system further comprises a third robotic arm. In
some
embodiments, the system further comprises a waste collecting vessel. In some
embodiments, the
system further comprises a HVAC system comprising a series of HEPA filters to
protect the
compound within the chamber. In some embodiments, a chamber, a storage vessel,
and a robotic
arm are configured respectively to transmit data.
[0009] In another aspect, the present disclosure provides a method for
producing a compound,
comprising: (a) providing a plurality of chambers, wherein a chamber of the
plurality of chambers
comprises a medium comprising at least one reagent; and (b) using at least one
computer processor
to generate instructions to: (i) fill a chamber with the medium; (ii) repeat
(i) until at least a portion
of the plurality of chambers is filled with the medium; (iii) synthesize the
compound in a chamber
that is filled with the medium and remove the compound from the chamber that
is filled with the
medium after a time interval Y; and (iv) continuously re-load, synthesize, and
remove the
compound. In some embodiments, the method further comprises rinsing or washing
the chamber
subsequent to (iii). In some embodiments, the method further comprises
providing a robotic arm
coupled to the at least one computer processor and configured to receive the
instructions in (b). In
some embodiments, the method further comprises filling a subsequent chamber
after a time interval
X. In some embodiments, the time interval Y is at least the time interval X
times the number of the
plurality of chambers. In some embodiments, the method further comprises the
at least one
computer processor to generate instructions to (1) fill a chamber with the
medium and remove the
medium comprising the compound and (2) repeat (1) in the same chamber until
receiving an input
for stop.
[0010] In some embodiments, the method further comprises filling a first
chamber with a first
medium comprising a first reagent and a second chamber with a second medium
comprising a
second reagent. In some embodiments, the first reagent is different from the
second reagent. In
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some embodiments, the first reagent is the same as the second reagent. In some
embodiments, the
first chamber and the second chamber comprise a substantially same
temperature. In some
embodiments, the first chamber and the second chamber comprise different
temperatures. In some
embodiments, the first chamber and the second chamber comprise a substantially
same reaction
time. In some embodiments, the first chamber and the second chamber comprise
different reaction
times. In some embodiments, the method further comprises monitoring a
temperature of the
medium. In some embodiments, the method further comprises monitoring a
temperature of an
atmosphere within a chamber. In some embodiments, the method further comprises
monitoring a
concentration of a molecule comprised within the medium. In some embodiments,
the method
further comprises monitoring an oxygen concentration of the atmosphere within
the chamber. In
some embodiments, the method further comprises monitoring a carbon dioxide
concentration of
the atmosphere within the chamber. In some embodiments, the method further
comprises
monitoring concentration of gas dissolved in the medium within the chamber. In
some
embodiments, the dissolved gas comprises carbon dioxide. In some embodiments,
the dissolved
gas comprises oxygen. In some embodiments, the method further comprises
monitoring a volume
level of a medium. In some embodiments, the method further comprises
monitoring a turbidity of
a medium. In some embodiments, the method further comprises monitoring a pH
value of a
medium. In some embodiments, the method further comprises monitoring a
humidity of the
atmosphere within the chamber. In some embodiments, the method further
comprises detecting a
contamination in a chamber. In some embodiments, the method further comprises
purifying the
compound.
[0011] In another aspect, the present disclosure provides a method for
producing a compound
comprising: (i) providing a plurality of chambers, (ii) filling a first
chamber with a medium,
wherein the medium comprises at least one reagent; (iii) filling a further
chamber after a time
interval X with the medium; (iv) repeating step (iii) until at least a portion
of the plurality of
chambers is filled with the medium; and (v) producing and removing the
compound after a time
interval Y from each of the portion of the plurality of chambers in step (iv),
thereby for a time
period of at least 2Y continuously producing the compound within the at least
a portion of the
plurality of chambers.
[0012] In some embodiments, the method further comprises rinsing or washing
the first chamber
subsequent to (v). In some embodiments, said filling is performed by at least
one robotic arm. In
some embodiments, the time interval Y is at least the time interval X times
the number of the
plurality of chambers. In some embodiments, the method further comprises
filling the first chamber
with a first medium comprising a first reagent and thefurther chamber with a
second medium
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comprising a second reagent. In some embodiments, the first reagent is
different from the second
reagent. In some embodiments, the first reagent is the same as the second
reagent. In some
embodiments, the first chamber and the further chamber comprise a
substantially same
temperature. In some embodiments, the first chamber and the further chamber
comprise different
temperatures. In some embodiments, the first chamber and the further chamber
comprise a
substantially same reaction time. In some embodiments, the first chamber and
the further chamber
comprise different reaction times. In some embodiments, the method further
comprises monitoring
a temperature of the medium. In some embodiments, the method further comprises
monitoring a
temperature of an atmosphere within the first chamber. In some embodiments,
the method further
comprises monitoring a concentration of a molecule comprised within the
medium. In some
embodiments, the method further comprises monitoring an oxygen concentration
of the
atmosphere within the first chamber. In some embodiments, the method further
comprises
monitoring a carbon dioxide concentration of the atmosphere within the first
chamber. In some
embodiments, the method further comprises monitoring a concentration of gas
dissolved in the
medium within the first chamber. In some embodiments, the method further
comprises monitoring
a volume level of the medium in the first chamber. In some embodiments, the
method further
comprises monitoring a turbidity of the medium. In some embodiments, the
method further
comprises monitoring a pH value of the medium. In some embodiments, the method
further
comprises monitoring a humidity of the atmosphere within the chamber. In some
embodiments,
the method further comprises detecting a contamination in a chamber of the
plurality of chambers.
In some embodiments, the method further comprises purifying the compound. In
some
embodiments, said compound is a nucleotide, such as RNA or DNA.
[0013] In another aspect, the present disclosure provides for a chamber for
the production of a
compound, the chamber comprising a bottom portion, a body portion comprising
an inner volume
configured to hold at least one reagent or a compound, an open end configured
to receive at least
one reagent, wherein at least part of said body portion comprises a flat
polygonal surface
configured to position and/or support a sensor in the reaction chamber. The
chamber of claim 121,
wherein the body portion comprises at least three flat polygonal surfaces.
[0014] In some embodiments, a cross-section of the body portion comprises a
polygonal shape. In
some embodiments, the polygonal shape comprises a trigonal, tetragonal,
pentagonal or hexagonal
shape. In some embodiments, the cross-section of the body portion is
hexagonal. In some
embodiments, the open end is configured to receive a removable lid for at
least partially closing
the open end. In some embodiments, the bottom is rounded. In some embodiments,
the chamber
comprises a chamber volume of about 0.1 mL to about 1000 mL. In some
embodiments, the
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chamber is configured for single use or multiple uses. In some embodiments,
the chamber further
comprises a scannable identification (ID) mechanism. In some embodiments, the
scannable ID
mechanism comprises an RFID.
[0015] In another aspect, the present disclosure provides for an assembly
comprising a lid
5 configured to at least partially close the open end of a chamber.
[0016] In some embodiments, the lid comprises a removable lid. In some
embodiments, the lid
comprises a push-on lid. In some embodiments, the lid comprises a screw lid.
In some
embodiments, the lid is glued to at least a portion of the chamber. In some
embodiments, the lid
comprises an opening. In some embodiments, the opening is centrally located
within the lid. In
10 some embodiments, the lid comprises a puncturable membrane. In some
embodiments, the
puncturable membrane comprises an elastic membrane.
[0017] In another aspect, the present disclosure provides for a cartridge
comprising a plurality of
chambers. In some embodiments, the plurality of chambers comprises between 2
and 20 chambers.
In some embodiments, the plurality of chambers comprises between 2 and 10
chambers. In some
embodiments, the cartridge further comprises at least one aperture. In some
embodiments, the at
least one aperture is configured to engage with a gripping mechanism. In some
embodiments, the
cartridge is configured to engage with a robotic arm and/or a handling device.
In some
embodiments, the robotic arm and/or the handling device comprises the gripping
mechanism. In
some embodiments, the cartridge comprises at least two apertures, wherein the
at least two
apertures are configured to engage with the gripping mechanism. In some
embodiments, the at
least one aperture is positioned at an upper surface of the cartridge. In some
embodiments, the
cartridge further comprises a scannable ID mechanism. In some embodiments, the
scannable ID
mechanism comprises the RFID.
[0018] In another aspect, the present disclosure provides a method comprising:
(a) providing or
obtaining the chamber as described herein; and producing a nucleic acid in the
chamber.
[0019] In some embodiments, the nucleic acid is a ribonucleic acid (RNA),In
some embodiments,
the chamber is comprised in an assembly as described herein. In some
embodiments, the chamber
is comprised in a cartridge as described herein.
[0020] In another aspect, the present disclosure provides a device for
producing a nucleic acid
compound, the device comprising: a plurality of chambers, wherein each chamber
of the plurality
of chambers is configured to contain at least one reagent for producing the
nucleic acid compound;
at least one robotic arm configured to load each chamber with the at least one
reagent; and at least
one computer processor operatively coupled to the plurality of chambers and
the at least one robotic
arm, wherein the at least one robotic arm is programmed to regularly fill the
plurality of chambers
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with at least one reagent after a time period Y, wherein the device is
programmed to remove nucleic
acid compound produced from each of the plurality of chambers after a time
interval Y, and
wherein the device is further programmed to re-load, produce and remove the
compound in a
continuous manner thereby for a time period of at least 2Y.
[0021] In another aspect, the present disclosure provides a device for
producing a nucleic acid
compound, the device comprising: a plurality of chambers, wherein each chamber
of the plurality
of chambers is configured to contain at least one reagent for producing the
nucleic acid compound
and the plurality of chambers is contained within a compartment capable of
moving said plurality
of chambers within the device; at least one robotic arm configured to load
each chamber with the
at least one reagent; and at least one computer processor operatively coupled
to the plurality of
chambers and the at least one robotic arm, wherein the at least one robotic
arm is programmed to
regularly fill the plurality of chambers with at least one reagent after a
time period Y, wherein the
device is programmed to remove nucleic acid compound produced from each of the
plurality of
chambers after a time interval Y.
[0022] In some embodiments, the device is further programmed to wash each of
the plurality of
chambers after removing the nucleic acid compound produced from each of the
plurality of
chambers after the time interval Y. In some embodiments, the robotic arm is
further programmed
to (1) fill each chamber with a medium comprising the at least one reagent and
remove the medium
comprising the nucleic acid compound and (2) repeat (1) in the same chamber
until receiving an
input for stop. In some embodiments, the device further comprises a second
plurality of chambers
configured to purify the nucleic acid compound. In some embodiments, the at
least one robotic arm
is further programmed to transport the nucleic acid compound to a chamber of
the second plurality
of chambers. In some embodiments, the device further comprises a second
robotic arm, wherein
the second robotic arm is programmed to transport the nucleic acid compound to
a chamber of the
second plurality of chambers. In some embodiments, the second plurality of
chambers is comprised
in a second compartment capable of moving the second plurality of chambers
within the device. In
some embodiments, the compartment comprises a conveyor or a carousel. In some
embodiments,
the second compartment comprises a second conveyor or carousel. In some
embodiments, a speed
of the compartment is determined at least in part by the time period Y.
[0023] Described herein are various embodiments of a chamber for the
production of a compound,
the chamber comprising a bottom portion, a body portion comprising an inner
volume configured
to hold at least one reagent or a compound, an open end configured to receive
at least one reagent,
wherein at least part of said body portion comprises a flat polygonal surface
configured to position
and/or support a sensor in the reaction chamber. In some embodiments, the body
portion comprises
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at least three flat polygonal surfaces. In some embodiments, a cross-section
of the body portion is
polygonal shape. In some embodiments, polygonal shape comprises a trigonal,
tetragonal,
pentagonal or hexagonal shape. In some embodiments, the cross-section of the
body portion is
hexagonal. In some embodiments, the open end is configured to receive a
removable lid for at least
partially closing the open end. In some embodiments, the bottom is rounded. In
some embodiments,
the chamber comprises a chamber volume of about 0.1 mL to about 1000 mL. In
some
embodiments, the chamber is configured for single use or multiple uses. The
chamber of any one
of the preceding claims, further comprising a scannable identification (ID)
mechanism. In some
embodiments, the scannable ID mechanism comprises an RFID.
[0024] Described herein are various embodiments of an assembly comprising a
lid configured to
at least partially close the open end of the chamber. In some embodiments, the
lid comprises a
removable lid. In some embodiments, the lid comprises a push-on lid. In some
embodiments, the
lid comprises a screw lid. In some embodiments, the lid is bonded (e.g.,
adhered) to another portion
of the assembly. In some embodiments, the lid is bonded to another portion of
the assembly with
use of an adhesive. In some embodiments, the lid is glued to another portion
of the assembly. In
some embodiments, the lid comprises an opening. In some embodiments, the
opening is centrally
located within the lid. In some embodiments, the lid comprises a puncturable
membrane. In some
embodiments, the puncturable membrane comprises an elastic membrane.
[0025] Described herein are various embodiments of a cartridge comprising a
plurality of
chambers. In some embodiments, the at least one aperture comprises between 2
and 20 chambers.
In some embodiments, the at least one aperture comprises between 2 and 10
chambers. In some
embodiments, the cartridge further comprises at least one aperture. In some
embodiments, the at
least one aperture is configured to engage with a gripping mechanism. In some
embodiments, the
cartridge is configured to engage with a robotic arm and/or a handling device.
In some
embodiments, the robotic arm and/or the handling device comprises the gripping
mechanism. In
some embodiments, the cartridge comprises at least two apertures, wherein the
at least two
apertures are configured to engage with the gripping mechanism. In some
embodiments, the at
least one aperture is positioned at an upper surface of the cartridge. The
cartridge of any one of the
preceding claims, further comprising the scannable ID mechanism. In some
embodiments, the
scannable ID mechanism comprises the RFID.
[0026] Described herein are various embodiments of methods and devices
comprising: providing
or obtaining the chamber as described in any one of the preceding claims; and
producing a nucleic
acid. In some embodiments, the nucleic acid is a ribonucleic acid (RNA). In
yet other
embodiments, the methods and devices as disclosed herein comprise a
hexagonally packed
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cartridge, the cartridge comprising: a plurality of hexagonal chambers, or
tubes, the hexagonal
chambers, or tubes configured for in vitro transcription (IVT) and down-stream
processing (DSP),
and wherein the cartridge is configured for each of the hexagonal chambers of
the plurality to be
arranged in a hexagonally-packed configuration. In some embodiments, the DSP
comprises post-
transcriptional capping and/or purification of the RNA molecule.
INCORPORATION BY REFERENCE
[0027] All publications, patents, and patent applications mentioned in this
specification are herein
incorporated by reference to the same extent as if each individual
publication, patent, or patent
application was specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] The novel features of the invention are set forth with particularity in
the appended claims.
A better understanding of the features and advantages of the present invention
will be obtained by
reference to the following detailed description that sets forth illustrative
embodiments, in which
the principles of the invention are utilized, and the accompanying drawings of
which:
[0029] FIG. 1 depicts an embodiment of the cartridge, chambers and robotic arm
of a system,
wherein the chambers are arranged in a geometrical configuration, for example
in a circular
manner, divided from each other by upstanding walls.
[0030] FIG. 2 depicts another embodiment of the cartridge, chambers and
robotic arm of a system,
wherein the chambers are arranged in a geometrical configuration, for example
in a rectangular
manner.
[0031] FIG. 3 depicts another embodiment of the cartridge, chambers and
robotic arm of a system,
wherein the chambers are arranged in a geometrical configuration, for example
in a honeycomb
structure.
[0032] FIG. 4 depicts a top view and movement of one embodiment of the
cartridge and chambers
of a system.
[0033] FIG. 5 depicts another embodiment of cartridges, chambers and robotic
arms of a system,
which allows to work with single and/or several cartridges in parallel.
[0034] FIG. 6 is a cross-sectional view of an embodiment depicting the
stackability of multiple
cartridges of a system in a vertical arrangement.
[0035] FIG. 7 depicts another embodiment of a plurality of chambers and
robotic arm of a system,
wherein chambers are configured to move on a conveyor belt powered by one or
more powered
pulleys.
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[0036] FIG. 8 depicts the combination of a cartridge, intermediate vessel and
down-stream
processing components of a system.
[0037] FIGS. 9A and 9B depicts a modular combination of components of a system
in accordance
with aspects of the present disclosure.
[0038] FIG. 10 depicts a computer system that is programmed or otherwise
configured to
implement methods provided herein.
[0039] FIG. 11 depicts a chamber in accordance with some embodiments of the
present
disclosure.
[0040] FIGS. 12A-12J depict various cartridges in accordance with some
embodiments of the
present disclosure.
[0041] FIG. 13 depicts an overview of an experiment.
[0042] FIG. 14 depicts an overview of an in vitro transcription (IVT) process
carried out as part
of an experiment described herein.
[0043] FIGS. 15A-15B show the results of quantitation of RNA synthesized in
accordance with
methods as disclosed herein.
[0044] FIGS. 16A-16B show the results of quantitation of residual nucleic
acids and protein in a
reactor as described herein.
[0045] FIG. 17 shows the proportion of double stranded RNA (dsRNA) to total
RNA in a sample
processed as part of an experiment described herein.
[0046] FIGS. 18A-18C depict results from computer simulations of systems and
methods
described herein.
DETAILED DESCRIPTION
[0047] There is need for the development of bioreactors which enable a
continuous manufacturing
process for pharmaceuticals, biochemical and biological compounds, including
biopharmaceuticals. Continuous manufacturing has many advantages: The
equipment is utilized
more efficiently as all unit operations are active at the same time, compared
with a batch process
which requires significant idle time of equipment. Less human intervention is
also contemplated,
reducing operational cost and human error. Continuous manufacturing may also
enable a smaller
footprint, potentially reducing operational costs. Lastly, pilot-scale
processes, which may also be
used for clinical supply, can transition into commercial manufacturing by
increasing the run time.
This eliminates scale-up and its associated construction and validation steps
and speeds up time to
market.
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[0048] Accordingly, disclosed herein are systems, methods and devices for the
continuous
production and processing of compounds, including biopharmaceutical compounds.
The system
and devices are operated in an automated manner and capable of operation under
Good
Manufacturing Practice (GMP)-compliant conditions.
5 [0049] Unless defined otherwise, all terms of art, notations and other
technical and scientific terms
or terminology used herein are intended to have the same meaning as is
commonly understood by
one of ordinary skill in the art to which the claimed subject matter pertains.
In some embodiments,
terms with commonly understood meanings are defined herein for clarity and/or
for ready
reference, and the inclusion of such definitions herein should not necessarily
be construed to
10 represent a substantial difference over what is generally understood in
the art.
[0050] Throughout this application, various embodiments may be presented in a
range format. It
should be understood that the description in range format is merely for
convenience and brevity
and should not be construed as an inflexible limitation on the scope of the
disclosure. Accordingly,
the description of a range should be considered to have specifically disclosed
all the possible
15 subranges as well as individual numerical values within that range. For
example, description of a
range such as from 1 to 6 should be considered to have specifically disclosed
subranges such as
from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6
etc., as well as individual
numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies
regardless of the breadth
of the range.
[0051] As used in the specification and claims, the singular forms "a", "an"
and "the" include
plural references unless the context clearly dictates otherwise. For example,
the term "a sample"
includes a plurality of samples, including mixtures thereof
[0052] The terms "determining," "measuring," "evaluating," "assessing,"
"assaying," and
"analyzing" are often used interchangeably herein to generally refer to forms
of measurement. The
terms include determining if an element is present or not (for example,
detection). These terms can
include quantitative, qualitative or quantitative and qualitative
determinations. Assessing can be
relative or absolute. "Detecting the presence of' can include determining the
amount of something
present in addition to determining whether it is present or absent depending
on the context.
[0053] The terms "subject," "individual," or "patient" are often used
interchangeably herein. A
"subject" can generally refer to a biological entity containing expressed
genetic materials. The
biological entity can be a plant, animal, or microorganism, including, for
example, bacteria,
viruses, fungi, and protozoa. The subject can be tissues, cells and their
progeny of a biological
entity obtained in vivo or cultured in vitro. The subject can be a mammal. The
mammal can be a
human. The subject may be diagnosed or suspected of being at high risk for a
disease. In some
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embodiments, the subject is not necessarily diagnosed or suspected of being at
high risk for the
disease.
[0054] The term "in vivo" is generally used to describe an event that takes
place in a subject's
body.
[0055] The term "ex vivo" is generally used to describe an event that takes
place outside of a
subject's body. An ex vivo assay is not performed on a subject. Rather, it is
performed upon a
sample separate from a subject. An example of an ex vivo assay performed on a
sample is an "in
vitro" assay.
[0056] The term "in vitro" is used to describe an event that takes places
contained in a container
for holding laboratory reagent such that it is separated from the biological
source from which the
material is obtained. In vitro assays can encompass cell-based assays in which
living or dead cells
are employed. In vitro assays can also encompass a cell-free assay in which no
intact cells are
employed.
[0057] As used herein, the term "about" a number generally refers to that
number plus or minus
10% of that number. The term "about" a range refers to that range minus 10% of
its lowest value
and plus 10% of its greatest value.
[0058] As used herein, the terms "therapy," or "therapeutic," or other derived
terms are generally
used in reference to a pharmaceutical or other intervention regimen for
obtaining beneficial or
desired results in the recipient. Beneficial or desired results include but
are not limited to a
therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit may
refer to eradication or
amelioration of symptoms or of an underlying disorder being treated. Also, a
therapeutic benefit
can be achieved with the eradication or amelioration of one or more of the
physiological symptoms
associated with the underlying disorder such that an improvement is observed
in the subject,
notwithstanding that the subject may still be afflicted with the underlying
disorder. A prophylactic
effect may include delaying, preventing, or eliminating the appearance of a
disease or condition,
delaying or eliminating the onset of symptoms of a disease or condition,
slowing, halting, or
reversing the progression of a disease or condition, or any combination
thereof. For prophylactic
benefit, a subject at risk of developing a particular disease, or to a subject
reporting one or more of
the physiological symptoms of a disease may undergo treatment, even though a
diagnosis of this
disease may not have been made.
[0059] Described herein are various embodiments of reactor systems. In some
embodiments, the
systems may comprise a chamber; a cartridge; a device for purification and/or
separation of a
compound; a lid; a sensor; or a combination thereof. In some embodiments, the
chamber may
comprise a bottom portion, a body portion comprising an inner volume, the
inner volume
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configured to hold at least one reagent or a compound. In some embodiments,
the chamber
comprises an open end configured to receive at least one reagent, wherein at
least part of the body
portion comprises a flat polygonal surface configured to allow positioning
and/or supporting a
sensor in the chamber. In some embodiments, at least one chamber is included
within a cartridge.
[0060] In some embodiments, various methods may comprise producing and/or
synthesizing
compounds in accordance with some embodiments. In some embodiments, the
compound may
comprise a chemical compound or biochemical compounds. In some embodiments,
various
methods may comprise manufacturing compound comprising a therapeutic, such as
a biological or
small molecule compound. In some embodiments, the compound may be an RNA
molecule,
obtained by an in vitro transcription method and further processed downstream,
for instance by
means of isolation, purification, and/or packaging.
[0061] Described herein are various embodiments of a reactor system
comprising: a chamber; and
a cartridge. In some embodiments, the chamber is configured for production of
a compound. In
some embodiments, the chamber comprises: a bottom portion; a body portion, the
body portion
comprising an inner volume configured to hold at least one reagent or a
compound; an open end
configured to receive at least one reagent, wherein at least part of the body
portion comprises a flat
polygonal surface configured to position and/or support a sensor in the
chamber. In some
embodiments, the body portion comprises at least three flat polygonal
surfaces. In some
embodiments, a cross-section of the body portion comprises a polygonal shape.
In some
embodiments, the polygonal shape comprises a trigonal, tetragonal, pentagonal
or hexagonal
shape. In some embodiments, the cross-section of the body portion comprises a
hexagonal shape.
In some embodiments, the open end is configured to receive a removable lid for
at least partially
closing the open end. In some embodiments, the bottom is rounded. In some
embodiments, the
chamber comprises a chamber volume of 0.1 mL to 1000 mL. In some embodiments,
the chamber
may be configured for single use or multiple uses. In some embodiments, the
system further
comprises a scannable identification mechanism. In some embodiments, the
scannable
identification mechanism comprises an RFID. In some embodiments, the chamber
comprises a lid
configured to at least partially close the open end of the chamber. In some
embodiments, the lid
comprises a removable lid. In some embodiments, the lid comprises a push-on
lid. In some
embodiments, the lid comprises a screw lid. In some embodiments, the lid is
bonded to at least part
of the chamber. In some embodiments, the lid is adhered to at least a part of
the chamber. In some
embodiments, the lid comprises a glued lid. In some embodiments, the lid
comprises an opening.
In some embodiments, the lid comprises the opening, wherein the opening is
centrally located
within the lid. In some embodiments, the lid comprises a puncturable membrane.
In some
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embodiments, the puncturable membrane comprises an elastic membrane. In some
embodiments,
the cartridge comprises a plurality of chambers. In some embodiments, the
cartridge comprises 2
to 20 chambers. In some embodiments, the cartridge comprises 2 to 10 chambers.
In some
embodiments, the cartridge comprises at least one aperture. In some
embodiments, the at least one
aperture is configured to engage with a gripping mechanism. In some
embodiments, the system
further comprises a robotic arm and/or a handling device. In some embodiments,
the robotic arm
and/or the handling device comprises the gripping mechanism. In some
embodiments, the cartridge
comprises at least two apertures, wherein the at least two apertures are
configured to engage with
the gripping mechanism. In some embodiments, the at least one aperture may be
positioned at an
upper surface of the cartridge. In some embodiments, the cartridge comprises
the scannable
identification mechanism. In some embodiments, the scannable identification
mechanism of the
cartridge comprises the RFID.
[0062] Described herein are various methods comprising: providing or obtaining
the system, in
accordance with some embodiments, and producing and/or synthesizing a nucleic
acid. In some
embodiments, the nucleic acid is a ribonucleic acid (RNA).
Compounds
[0063] Provided herein is a system for producing a compound, including drug
substance, active
pharmaceutical ingredient and/or biopharmaceutical compounds, comprising a
plurality of
chambers, at least one robotic arm and at least one computer processor
operatively coupled to the
plurality of chambers and the at least one robotic arm. In some embodiments,
each chamber of the
plurality of chambers is configured to contain at least one reagent for
producing the compound. In
further embodiments, the at least one robotic arm is configured to load each
chamber with the at
least one reagent.
[0064] In some embodiments described herein, the at least one robotic arm is
programmable to (i)
fill a first chamber with the at least one reagent; (ii) fill a second chamber
after a time interval X
with the at least one reagent; and (iii) repeat step (ii) until at least a
portion of the plurality of
chambers is filled with the at least one reagent. In some embodiments, the
compound is produced
and removed after a time interval Y from each of the portions of plurality of
chambers in step (iii).
In some embodiments, the system is programmable in at least a portion of the
plurality of chambers
to re-load, produce and remove the compound as in steps (i)-(iii) thereby for
a time period of at
least 2Y continuously producing the compound within at least a portion of the
plurality of the
chambers.
[0065] In some embodiments, the compound is continuously produced for a time
period of at least
3Y within at least a portion of the plurality of chambers. In yet other
embodiments, the compound
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is continuously produced for a time period of at least 4Y within at least a
portion of the plurality
of chambers. In still other embodiments, the compound is continuously produced
for a time period
of at least 5Y within at least a portion of the plurality of chambers. In
still other embodiments, the
compound is continuously produced for a time period of at least more than 5Y
within at least a
portion of the plurality of chambers. In some embodiments, the compound is
continuously
produced for a time period of at least 6Y within at least a portion of the
plurality of chambers. In
some embodiments, the compound is continuously produced for a time period of
at least 7Y within
at least a portion of the plurality of chambers. In some embodiments, the
compound is continuously
produced for a time period of at least 8Y within at least a portion of the
plurality of chambers. In
.. some embodiments, the compound is continuously produced for a time period
of at least 9Y within
at least a portion of the plurality of chambers. In some embodiments, the
compound is continuously
produced for a time period of at least 10Y within at least a portion of the
plurality of chambers. In
some embodiments, the compound is continuously produced for a time period of
at least 20Y
within at least a portion of the plurality of chambers. In some embodiments,
the compound is
continuously produced for a time period of at least 30Y within at least a
portion of the plurality of
chambers. In some embodiments, the compound is continuously produced for a
time period of at
least 40Y within at least a portion of the plurality of chambers. In some
embodiments, the
compound is continuously produced for a time period of at least 50Y within at
least a portion of
the plurality of chambers. In some embodiments, the compound is continuously
produced for a
time period of at least 60Y within at least a portion of the plurality of
chambers. In some
embodiments, the compound is continuously produced for a time period of at
least 70Y within at
least a portion of the plurality of chambers. In some embodiments, the
compound is continuously
produced for a time period of at least 80Y within at least a portion of the
plurality of chambers. In
some embodiments, the compound is continuously produced for a time period of
at least 90Y
within at least a portion of the plurality of chambers. In some embodiments,
the compound is
continuously produced for a time period of at least 100Y within at least a
portion of the plurality
of chambers. In some embodiments, the compound is continuously produced for a
time period of
at least 200Y within at least a portion of the plurality of chambers. In some
embodiments, the
compound is continuously produced for a time period of at least 300Y within at
least a portion of
the plurality of chambers. In some embodiments, the compound is continuously
produced for a
time period of at least 400Y within at least a portion of the plurality of
chambers. In some
embodiments, the compound is continuously produced for a time period of at
least 500Y within at
least a portion of the plurality of chambers.
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[0066] In some embodiments described herein, the compound, including
biopharmaceutical
compounds, comprises nucleic acids including DNA and RNA, modified DNA and
RNA,
polypeptides, proteins, and modified proteins.
[0067] In some embodiments, the biochemical compound comprises RNA molecules
that can be
5 used in current and/or potential RNA-based therapies. In some
embodiments, the biochemical
compound comprises long-chain RNA, coding RNA, non-coding RNA, long non-coding
RNA,
single stranded RNA (ssRNA), double stranded RNA (dsRNA), linear RNA (linRNA),
circular
RNA (circRNA), messenger RNA (mRNA), self-amplifying mRNA (SAM), Trans
amplifying
mRNA, RNA oligonucleotides, antisense oligonucleotides, small interfering RNA
(siRNA), small
10 hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR/Cas9 guide RNAs,
riboswitches,
immunostimulating RNA (isRNA), ribozymes, aptamers, ribosomal RNA (rRNA),
transfer RNA
(tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, small nuclear RNA
(snRNA), small
nucleolar RNA (snoRNA), microRNA (miRNA), and a Piwi-interacting RNA (piRNA).
[0068] In some embodiments, the biochemical compound comprises modified RNA
molecules. In
15 some embodiments, the modification of RNA molecule comprises chemical
modifications
comprising backbone modifications as well as sugar modifications or base
modifications. In this
context, a modified RNA molecule as defined herein comprises nucleotide
analogues/modifications, e.g. backbone modifications, sugar modifications or
base modifications.
A backbone modification in connection with the present disclosure is a
modification, in which
20 phosphates of the backbone of the nucleotides contained in an RNA
molecule are chemically
modified. A sugar modification in connection with the present disclosure is a
chemical
modification of the sugar of the nucleotides of the RNA molecule. Furthermore,
a base
modification in connection with the present disclosure is a chemical
modification of the base
moiety of the nucleotides of the RNA molecule. In this context, nucleotide
analogues or
modifications are selected from nucleotide analogues, which are applicable for
transcription and/or
translation. In further embodiments, the modified RNA comprises nucleoside
modifications
selected from 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, pseudo-iso-
cytidine, 5-aminoallyl-
uridine, 5-iodo-uridine, Ni -methyl-pseudouridine, 5,6-dihydrouridine, a-thio-
uridine, 4-thio-
uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine,
pyrrolo-cytidine,
inosine, a-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-
guanosine, 7-deaza-
guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-
purine, pseudo-
iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, a-thio-adenosine, 8-azido-
adenosine, 7-
deaza-adenosine.
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[0069] In some embodiments, the biochemical compound comprises protein
molecules that can be
used in current and/or potential protein-based therapies. In some embodiments,
the biochemical
compound comprises antibody-based drugs, glycoconjugates, Fc fusion proteins,
anticoagulants,
blood factors, bone morphogenetic proteins, engineered protein scaffolds,
enzymes, growth
factors, hormones, interferons, interleukins and other cytokines, viral (and
other pathogen) proteins
(or antigens), parts of proteins (truncated proteins, ectodomains, stem
domains, etc.) or chimeric
proteins and thrombolytics. In some embodiments, the biochemical compound
comprises modified
protein molecules that can be used in protein-based therapies. In some
embodiments, the
modification of protein molecule comprises phosphorylation, glycosylation,
acetylation,
ubiquitylation/sumoylation, methylation, palmitoylation, quinone, amidation,
myristoylation,
pyrrolidone carboxylic acid, hydroxylation, phosphopantetheine, prenylation,
GPI anchoring,
oxidation, ADP-ribosylation, sulfation, S-nitrosylation, citrullination,
nitration, gamma-
carboxyglutamic acid, formylation, hypusine, topaquinone /TPQ, bromination,
lysine
topaquinone/LTQ, tryptophan tryptophylquinone/TTQ, iodination, pegylation and
cysteine
tryptophylquinone / CTQ.
Compound production
[0070] In some embodiments of the present disclosure, the chamber is designed
to accommodate
a reaction/process or part of a reaction/process taking place in the chamber.
In some embodiments,
the reaction relates to in vitro transcription of RNA from a DNA template
and/or in vitro (cell-free)
translation of RNA to protein and/or a combination of both processes, i.e.,
from DNA to RNA
through transcription and from RNA to protein through translation. In some
embodiments, the
reaction may also pertain to the culturing and growing of cells and expressing
proteins or viral
particles. Not meant to be limiting, in further embodiments, the reaction may
also relate to any
incubation steps following a given temperature and time profile.
[0071] In some embodiments, the chamber is configured to conduct an in vitro
transcription
reaction. In some embodiments, the chamber is configured to conduct an in
vitro translation. In
some embodiments, the in vitro translation is cell dependent. In some
embodiments, the in vitro
translation is cell free. In some embodiments, the chamber is not designed to
culture a living cell.
[0072] In some embodiments, the in vitro transcription relates to a process in
which RNA is
synthesized in a cell-free system (in vitro). In some embodiments, cloning
vector(s) DNA,
particularly plasmid DNA vectors are applied as template for the generation of
RNA transcripts
following linearization of circular plasmid DNA molecule. These cloning
vectors are generally
designated as transcription vector. RNA may be obtained by DNA dependent in
vitro transcription
of an appropriate DNA template. A promoter for controlling RNA in vitro
transcription can be any
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promoter for any DNA dependent RNA polymerase. In some embodiments, a viral
promoter binds
a viral RNA polymerase and is at least one promoter selected from the list
consisting of T7, T3,
T7lac, SP6, pL, CMV, SV40, and CaMV35S. Alternately or in combination, the
nucleic acid
fragment comprising promoter sequence comprises a bacterial promoter. In some
embodiments,
the bacterial promoter binds a bacterial RNA polymerase and is at least one
promoter selected from
the list consisting of araBAD, trp, lac, and Ptac. Sometimes, the nucleic acid
fragment comprising
promoter sequence comprises a eukaryotic promoter. In some embodiments, the
eukaryotic
promoter binds a eukaryotic RNA polymerase and is at least one promoter
selected from the list
consisting of EFla, PGK1, Ubc, beta actin, CAG, TRE, UAS, Ac5, Polyhedrin,
CaMKIIa, ALB,
GAL1, GAL10, TEF1, GDS, ADH1, Ubi, H1, and U6. In some embodiments, the
eukaryotic
promoter is at least one promoter selected from the list consisting of an RNA
pol I promoter, an
RNA pol II promoter and an RNA pol III promoter.
[0073] In some embodiments, the DNA dependent RNA polymerases comprise at
least one of a
T7 RNA polymerase, a T3 RNA polymerase, a 5P6 RNA polymerase, a RNA polymerase
I, a RNA
polymerase II, a RNA polymerase III, a RNA polymerase IV, a RNA polymerase V,
and a single
subunit RNA polymerase. The DNA template for RNA in vitro RNA transcription
may be obtained
by cloning of a nucleic acid, in particular cDNA corresponding to the
respective RNA to be in vitro
transcribed, and introducing it into an appropriate vector for RNA in vitro
transcription, for
example in plasmid circular plasmid DNA. The cDNA may be obtained by reverse
transcription
of mRNA or chemical synthesis. Moreover, the DNA template for in vitro RNA
synthesis may
also be obtained by gene synthesis.
[0074] In some embodiments, the DNA template relates to a DNA molecule
comprising a nucleic
acid sequence encoding the RNA sequence. The template DNA is used as a
template for RNA in
vitro transcription in order to produce the RNA encoded by the template DNA.
Therefore, the
template DNA comprises all elements necessary for RNA in vitro transcription,
particularly a
promoter element for binding of a DNA dependent RNA polymerase as e.g. T3, T7
and 5P6 RNA
polymerases 5' of the DNA sequence encoding the target RNA sequence. The
poly(A) tail can be
either encoded into the DNA template or added enzymatically to RNA in a
separate step after in
vitro transcription. In some embodiments, the template DNA comprises primer
binding sites 5'
and/or 3' of the DNA sequence encoding the target RNA sequence to determine
the identity of the
DNA sequence encoding the target RNA sequence e.g. by PCR or DNA sequencing.
In some
embodiments, the DNA template comprises a DNA vector, such as a plasmid DNA,
which
comprises a nucleic acid sequence encoding the RNA sequence. In some
embodiments, the DNA
template comprises a linear or a circular DNA molecule.
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[0075] In some embodiments of the present invention, a DNA template encodes a
different RNA
molecule species. In some embodiments the DNA template contains a sub-genomic
promoter and
a large ORF encoding for non-structural proteins which, following delivery of
the
biopharmaceutical into the cytosol, are transcribed in four functional
components (nsP 1, nsP2,
nsP3, and nsp4) by the encoded RNA-dependent RNA polymerase (RDRP). RDRP than
produces
a negative-sense copy of the genome which serves as a template for two
positive-strand RNA
molecules: the genomic mRNA and a shorter sub-genomic mRNA. This sub-genomic
mRNA is
transcribed at very high levels, allowing the amplification of mRNA encoding
the antigen of
choice. A different RNA molecule species may encode an antigen of different
serotypes or strains
of a pathogen, a different allergen, a different autoimmune antigen, a
different antigen of a
pathogen, different adjuvant proteins, a different isoform or variant of a
cancer or tumor antigen,
a different tumor antigen of one patient, one antibody among a group of
antibodies which target
different epitopes of a protein or of a group of proteins, different proteins
of a metabolic pathway,
a single protein among a group of proteins which are defect in a subject, or a
different isoform of
a protein for molecular therapy.
[0076] In some embodiments, the pathogen is selected from the group consisting
of a virus,
bacterium, prion, fungus, protozoon, viroid, and parasite.
[0077] In some embodiments, the pathogen is selected from the group that
causes human disease
which includes but are not limited to, Bacillus anthracis (anthrax),
Clostridium botulinum toxin
(botulism), Yersinia pestis (plague). Variola major (smallpox) and other
related pox viruses,
Francisella tularensis (tularemia), Viral hemorrhagic fevers, Arenaviruses,
(e.g., Junin, Machupo,
Guanarito, Chapare, Lassa, and/or Lujo), Bunyaviruses (e.g., Hantaviruses
causing Hanta
Pulmonary syndrome, Rift Valley Fever, and/or Crimean Congo Hemorrhagic
Fever),
Flaviviruses, Dengue, Filoviruses (e.g., Ebola and Marburg viruses),
Burkholderia pseudomallei
(melioidosis), Coxiella burnetii (Q fever), Brucella species (brucellosis),
Burkholderia mallei
(glanders), Chlamydia psittaci (Psittacosis), Ricin toxin (Ricinus communis),
Epsilon toxin
(Clostridium perfringens), Staphylococcus enterotoxin B (SEB), Typhus fever
(Rickettsia
prowazekii), Food- and waterborne pathogens, Diarrheagenic E. coli, Pathogenic
Vibrios, Shigella
species, Salmonella, Listeria monocytogenes, Campylobacter jejuni, Yersinia
enterocolitica,
Caliciviruses, Hepatitis A, Cryptosporidium parvum, Cyclospora cayatanensis,
Giardia lamblia,
Entamoeba histolytica , Toxoplasma gondii, Naegleria fowleri, Balamuthia
mandrillaris, Fungi,
Microsporidia, Mosquito-borne viruses (e.g., West Nile virus (WNV), LaCrosse
encephalitis
(LACV), California encephalitis, Venezuelan equine encephalitis (VEE), Eastern
equine
encephalitis (EEE), Western equine encephalitis (WEE), Japanese encephalitis
virus (JE), St. Louis
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encephalitis virus (SLEV), Yellow fever virus (YFV), Chikungunya virus, Zika
virus, Nipah and
Hendra viruses, Additional hantaviruses, Tickborne hemorrhagic fever viruses,
Bunyaviruses,
Severe Fever with Thrombocytopenia Syndrome virus (SFTSV), Heartland virus,
Flaviviruses
(e.g., Omsk Hemorrhagic Fever virus, Alkhurma virus, Kyasanur Forest virus),
Tickborne
encephalitis complex flaviviruses, Tickborne encephalitis viruses,
Powassan/Deer Tick virus,
Tuberculosis, including drug-resistant Tuberculosis, Influenza virus, Prions,
Streptococcus,
Pseudomonas, Shigella, Campylobacter, Salmonella, Clostridium, Escherichia,
Hepatitis C,
papillomavirus, Epstein-Barr virus, varicella, variola, Orthomyxovirus, Severe
acute respiratory
syndrome associated coronavirus (SARS-CoV), SARS-CoV-2 (COVID-19), MERS-CoV,
other
highly pathogenic human coronaviruses, or any combination thereof.
[0078] In some embodiments, the virus is a respiratory virus that primarily
results in respiratory
symptoms including, without limitation, coronaviruses, influenza viruses,
adenoviruses,
rhinoviruses, coxsackieviruses, and metapneumoviruseses. In some embodiments,
the virus is an
enteric virus that primarily results in digestive symptoms including, without
limitation,
enteroviruses, noroviruses, heptoviruses, reoviruses, rotaviruses,
parvoviruses, toroviruses, and
mastadenovirus. In certain embodiments, the virus is a hemorrhagic fever virus
including, without
limitation, Ebola virus, Marburg virus, dengue fever virus, yellow fever
virus, Rift valley fever
virus, hanta virus, and Lassa fever virus.
[0079] In some embodiments, a DNA template encodes a different RNA molecule
species. In some
embodiments, different RNA molecule species comprises RNA constructs
representing an
engineered (non-natural) variant of a protein (e.g., chimeric protein), or
fragment thereof. In some
embodiments, a DNA template encodes different whole proteins that are
separated by a linker. In
some embodiments, different RNA molecule species encodes a different
pathogenic antigen or a
fragment or variant thereof. In some embodiments, the pathogen-associated
antigen is from an
influenza virus. In some embodiments, the pathogen-associated antigen is from
an influenza A
virus, such as the H5N1 strain. In some embodiments, the pathogen-associated
antigen is from an
influenza B virus. In some embodiments, the pathogen-associated antigen is an
influenza matrix
M1 protein or a fragment thereof. In certain embodiments, the pathogen-
associated antigen is an
influenza neuraminidase or a fragment thereof. In certain embodiments, the
pathogen-associated
antigen is an influenza hemagglutinin or a fragment thereof For example, the
pathogen-associated
antigen may comprise an entire hemagglutinin, an HAI domain, an HA2 domain or
any antigenic
portion thereof.
[0080] In some embodiments, the pathogen-associated antigen is a Coronaviridae
antigen. In some
embodiments, the Coronaviridae exhibits human tropism. In some embodiments,
the
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Coronaviridae is selected from the list consisting of SARS Coronavirus (SARS-
CoV-1), COVID-
19 (SARS-CoV-2), MERS-coronavirus (MERS-CoV), or any combination thereof. In
some
embodiments, the Coronaviridae comprises SARS Coronavirus (SARS-CoV-1). In
some
embodiments, the Coronaviridae comprises COVID-19 (SARS-CoV-2). In some
embodiments,
5 the Coronaviridae comprises MERS-coronavirus (MERS-CoV). In some
embodiments, the
Coronaviridae antigen comprises a spike protein, an envelope protein, a
nucleocapsid protein, a
membrane protein, a membrane glycoprotein, or a non-structural protein. In
some embodiments,
the Coronaviridae antigen comprises a spike protein, an envelope small
membrane protein, a
membrane protein, a non-structural protein 6 (NSP6), a nucleoprotein, an ORF10
protein, Protein
10 3a, Protein7a, Protein 9b, structural protein 8, uncharacterized protein
4, or any combination
thereof.
[0081] In some embodiments, a DNA template encodes a different RNA molecule
species. In some
embodiments, a different RNA molecule species encodes a different cancer or
tumor antigen, or a
fragment or variant thereof, selected from the group consisting of caTRL4,
CD4OL, CD34, CD41,
15 G6B, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9,
GP1BA, DSC2,
FCGR2A, TNFRSF10A, TNFRSF10B, TM4SF1, Her2, Trop2, CEA, NaPi2b, uPAR, CDCP1,
MUC-1, MUC-16, CEACAM-5, MR-1, Fn14, MAGE-3, NY-ESO-1, EGFR, PDGFR, IGF1R,
CSF-1R, PSMA, PSCA, STEAP-1, FAP, TEM8, 5T4, VEGFR, NRP1, CD19, CD20, CD22,
CD25, CD30, CD33, CD37, CD38, CD39, CD44, CD47, CD52, CD70, CD71, CD74, CD79b,
20 CD132, CD133, CD138, CD166, CD205, CD276, ROR1, ROR2, Glypican 3, Trail
Receptor 2
(DRS), PD-L1, Mesothein, Bombesin, EpCAM, DARPP, CSPG4, Galectin-3, Integrin
avI31,
Integrin avI33, Integrin avI35, Integrin avI36, Integrin a5131, Integrin alpha-
3, Integrin alpha-5,
Integrin beta-6, Nectin-4, DLL3, Transferrin Receptor, Folate Receptor alpha,
Tissue Factor,
BCMA, c-Met, LIV-1, AXL, AFP, ENPP3, CLDN6/9, DPEP3, RNF43, LRRC15, PTK7, P-
25 cadherin, FLT3, EphA2, MTI-MMP, CXCR6, GD2, or Smoothened antigen (Smo).
[0082] Components of the System
[0083] Described herein are various embodiments, of a system comprising a
chamber; a cartridge;
a separation and/or purification device; a lid; a sensor; an identification
(ID) mechanism; or a
combination thereof.
[0084] Chambers & Cartridge
[0085] Described herein are various embodiments of a chamber for the
production of a compound,
the chamber comprising a bottom portion, a body portion comprising an inner
volume configured
to hold at least one reagent or a compound, an open end configured to receive
at least one reagent,
wherein at least part of said body portion comprises a flat polygonal surface
configured to position
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and/or support a sensor in the reaction chamber. In some embodiments, the body
portion comprises
at least three flat polygonal surfaces. In some embodiments, a cross-section
of the body portion is
polygonal shape. In some embodiments, polygonal shape comprises a trigonal,
tetragonal,
pentagonal or hexagonal shape. In some embodiments, the cross-section of the
body portion is
.. hexagonal. In some embodiments, the open end is configured to receive a
removable lid for at least
partially closing the open end. In some embodiments, the bottom is rounded. In
some embodiments,
the chamber comprises a chamber volume of about 0.1 mL to about 1000 mL. In
some
embodiments, the chamber is configured for single use or multiple uses. The
chamber of any one
of the preceding claims, further comprising a scannable identification (ID)
mechanism. In some
embodiments, the scannable ID mechanism comprises an RFID.
[0086] Described herein are various embodiments of an assembly comprising a
lid configured to
at least partially close the open end of the chamber. In some embodiments, the
lid comprises a
removable lid. In some embodiments, the lid comprises a push-on lid. In some
embodiments, the
lid comprises a screw lid. In some embodiments, the lid comprises a non-
removable lid. In some
.. embodiments, the lid is bonded to at least a part of the chamber. In some
embodiments, the lid is
adhered to at least a part of the chamber. In some embodiments, the lid is
glued to at least part of
the chamber. In some embodiments, the lid comprises an opening. In some
embodiments, the
opening is centrally located within the lid. In some embodiments, the lid
comprises a puncturable
membrane. In some embodiments, the puncturable membrane comprises an elastic
membrane.
[0087] Described herein are various embodiments of a cartridge comprising a
plurality of
chambers. In some embodiments, the at least one aperture comprises between 2
and 20 chambers.
In some embodiments, the at least one aperture comprises between 2 and 10
chambers. In some
embodiments, the cartridge further comprises at least one aperture. In some
embodiments, the at
least one aperture is configured to engage with a gripping mechanism. In some
embodiments, the
cartridge is configured to engage with a robotic arm and/or a handling device.
In some
embodiments, the robotic arm and/or the handling device comprises the gripping
mechanism. In
some embodiments, the cartridge comprises at least two apertures, wherein the
at least two
apertures are configured to engage with the gripping mechanism. In some
embodiments, the at
least one aperture is positioned at an upper surface of the cartridge. The
cartridge of any one of the
preceding claims, further comprising the scannable ID mechanism. In some
embodiments, the
scannable ID mechanism comprises the RFID.
[0088] In some embodiments, the plurality of chambers is housed in a
cartridge.
[0089] In some embodiments, the cartridge comprises at least 2 chambers, 3
chambers, 4
chambers, 5 chambers, 6 chambers, 7 chambers, 8 chambers, 9 chambers, 10
chambers, 11
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chambers, 12 chambers, 13 chambers, 14 chambers, 15 chambers, 16 chambers, 17
chambers, 18
chambers, 19 chambers, 20 chambers, 21 chambers, 22 chambers, 23 chambers, 24
chambers, 25
chambers, 26 chambers, 27 chambers, 28 chambers, 29 chambers, 30 chambers, 31
chambers, 32
chambers, 33 chambers, 34 chambers, 35 chambers, 36 chambers, 37 chambers, 38
chambers, 39
chambers, 40 chambers, 41 chambers, 42 chambers, 43 chambers, 44 chambers, 45
chambers, 46
chambers, 47 chambers, 48 chambers, 49 chambers, or 50 chambers. In some
embodiments, the
cartridge comprises at least about 60 chambers, about 70 chambers, about 80
chambers, about 90
chambers, about 100 chambers, about 110 chambers, about 120 chambers, about
130 chambers,
about 140 chambers, about 150 chambers, about 160 chambers, about 170
chambers, about 180
chambers, about 190 chambers, or about 200 chambers.
[0090] In some embodiments, the cartridge comprises 2 to 20 chambers. In some
embodiments,
the cartridge comprises 2 to 10 chambers. In some embodiments, the cartridge
comprises at least
about 2 chambers. In some embodiments, the cartridge comprises at least about
12 chambers. In
some embodiments, the cartridge comprises at least about 24 chambers. In some
embodiments, the
cartridge comprises at least about 36 chambers. In some embodiments, the
cartridge comprises at
least about 48 chambers.
In some embodiments, the chamber is removable from the cartridge. In some
embodiments, the
chamber is not removable from the cartridge.
[0091] In some embodiments, the chamber comprises a volume of at least about
0.1 mL to 1000
mL. In some embodiments, the chamber comprises a volume of at least about 0.1
mL, about 0.3
mL, about 0.5 mL, about 1 mL, about 1.5 mL, about 2 mL, about 2.5 mL, about 3
mL, about 4 mL,
about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about
12 mL, about
15 mL, about 17 mL, about 20 mL, about 25 mL, about 30 mL, about 35 mL, about
40 mL, about
45 mL, about 50 mL, about 55 mL, about 60 mL, about 65 mL, about 70 mL, about
75 mL, about
80 mL, about 85 mL, about 90 mL, about 95 mL, about 100 mL, about 300 mL,
about 500 mL,
about 1000 mL, or more. In some embodiments, the chamber comprises a volume of
not more than
about 1000 mL, not more than about 500 mL, not more than about 300 mL, not
more than about100
mL, not more than about 95 mL, not more than about 90 mL, not more than about
85 mL, not more
than about 80 mL, not more than about 75 mL, not more than about 70 mL, not
more than about
65 mL, not more than about 60 mL, not more than about 55 mL, not more than
about 50 mL, not
more than about 45 mL, not more than about 40 mL, not more than about 35 mL,
not more than
about 30 mL, not more than about 25 mL, not more than about 20 mL, not more
than about 15 mL,
not more than about 10 mL, not more than about 9 mL, not more than about 8 mL,
not more than
about 7 mL, not more than about 6 mL, not more than about 5 mL, not more than
about 4 mL, not
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more than about 3 mL, not more than about 2 mL, not more than about 1 mL, not
more than about
0.5 mL, not more than about 0.3 mL, or not more than about 0.1 mL. In some
embodiments, the
chamber comprises a volume of between about 1 mL to about 100 mL, between
about 10 mL to
90 mL, between about 15 mL to about 80 mL, between about 20 mL to about 70 mL,
between
about 25 mL to about 60 mL or between about 30 mL to about 50 mL.
[0092] In some embodiments, the chamber comprises a volume of at least about
150 mL, about
200 mL, about 250 mL, about 300 mL, about 350 mL, about 400 mL, about 450 mL,
about 500
mL, about 550 mL, about 600 mL, about 650 mL, about 700 mL, about 750 mL,
about 800 mL,
about 850 mL, about 900 mL, about 950 mL, about 1000 mL, about 2000 mL, about
3000 mL,
about 4000 mL, about 5000 mL, about 6000 mL, about 7000 mL, about 8000 mL,
about 9000 mL,
about 10000 mL, about 15000 mL, about 20000 mL, about 25000 mL, about 30000
mL, about
35000 mL, about 40000 mL, about 45000 mL, or about 50000 mL. In some
embodiments, the
chamber comprises a volume of not more than about 50000 mL, not more than
about 45000 mL,
not more than about 40000 mL, not more than about 35000 mL, not more than
about 30000 mL,
not more than about 25000 mL, not more than about 20000 mL, not more than
about 15000 mL,
not more than about 10000 mL, not more than about 9000 mL, not more than about
8000 mL, not
more than about 7000 mL, not more than about 6000 mL, not more than about 5000
mL, not more
than about 4000 mL, not more than about 3000 mL, not more than about 2000 mL,
not more than
about 1000 mL, not more than about 950 mL, not more than about 900 mL, not
more than about
850 mL, not more than about 800 mL, not more than about 750 mL, not more than
about 700 mL,
not more than about 650 mL, not more than about 600 mL, not more than about
550 mL, not more
than about 500 mL, not more than about 450 mL, not more than about 400 mL, not
more than about
350 mL, not more than about 300 mL, not more than about 250 mL, not more than
about 200 mL,
not more than about 150 mL. In some embodiments, the chamber comprises a
volume of between
about 150 mL to about 50000 mL, between about 200 mL to 45000 mL, between
about 250 mL to
about 40000 mL, between about 300 mL to about 35000 mL, between about 350 mL
to about
30000 mL, between about 400 mL to about 25000 mL, between about 450 mL to
about 20000 mL,
between about 500 mL to about 15000 mL. between about 550 mL to about 10000
mL, between
about 600 mL to about 9000 mL, between about 650 mL to 8000 mL, between about
700 mL to
about 7000 mL, between about 750 mL to about 6000 mL, between about 800 mL to
about 5000
mL, between about 850 mL to about 4000 mL, between about 900 mL to about 3000
mL, between
about 950 mL to about 2000 mL or between about 1000 mL to about 1500 mL.
[0093] In some embodiments described herein, a number of the plurality of
chambers is at least
the time interval Y divided by the time interval X. In some embodiments, a
number of the plurality
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of chambers is less than the time interval Y divided by the time interval X.
In some embodiments,
a number of the plurality of chambers is greater than the time interval Y
divided by the time interval
X. In some embodiments, when the last chamber of the plurality of chambers is
filled with the at
least one reagent, the compound is produced and removed from the first chamber
of the plurality
of chambers. In some embodiments, the time interval Y is at least the time
interval X times the
number of the plurality of chambers. In some embodiments, for a time period of
at least 2Y, the
system continuously produces the compound within the plurality of the
chambers. In some
embodiments, the compound is continuously produced for a time period of at
least 3Y within at
least a portion of the plurality of chambers. In yet other embodiments, the
compound is
continuously produced for a time period of at least 4Y within at least a
portion of the plurality of
chambers. In still other embodiments, the compound is continuously produced
for a time period of
at least 5Y within at least a portion of the plurality of chambers. In still
other embodiments, the
compound is continuously produced for a time period of at least 6Y within at
least a portion of the
plurality of chambers. In still other embodiments, the compound is
continuously produced for a
time period of at least 7Y within at least a portion of the plurality of
chambers. In still other
embodiments, the compound is continuously produced for a time period of at
least 8Y within at
least a portion of the plurality of chambers. In still other embodiments, the
compound is
continuously produced for a time period of at least 9Y within at least a
portion of the plurality of
chambers. In still other embodiments, the compound is continuously produced
for a time period of
at least 10Y within at least a portion of the plurality of chambers. In still
other embodiments, the
compound is continuously produced for a time period of at least 20Y within at
least a portion of
the plurality of chambers. In still other embodiments, the compound is
continuously produced for
a time period of at least 30Y within at least a portion of the plurality of
chambers. In still other
embodiments, the compound is continuously produced for a time period of at
least 40Y within at
least a portion of the plurality of chambers. In still other embodiments, the
compound is
continuously produced for a time period of at least 50Y within at least a
portion of the plurality of
chambers. In still other embodiments, the compound is continuously produced
for a time period of
at least 60Y within at least a portion of the plurality of chambers. In still
other embodiments, the
compound is continuously produced for a time period of at least 70Y within at
least a portion of
the plurality of chambers. In still other embodiments, the compound is
continuously produced for
a time period of at least 80Y within at least a portion of the plurality of
chambers. In still other
embodiments, the compound is continuously produced for a time period of at
least 90Y within at
least a portion of the plurality of chambers. In still other embodiments, the
compound is
continuously produced for a time period of at least 100Y within at least a
portion of the plurality
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of chambers. In still other embodiments, the compound is continuously produced
for a time period
of at least 200Y within at least a portion of the plurality of chambers. In
still other embodiments,
the compound is continuously produced for a time period of at least 300Y
within at least a portion
of the plurality of chambers. In still other embodiments, the compound is
continuously produced
5 for a time period of at least 400Y within at least a portion of the
plurality of chambers. In still other
embodiments, the compound is continuously produced for a time period of at
least 500Y within at
least a portion of the plurality of chambers. In still other embodiments, the
compound is
continuously produced for a time period of at least more than 5Y within at
least a portion of the
plurality of chambers.
10 [0094] In some embodiments, the time interval X comprises time at least
about 0.1 minute, about
0.2 minutes, about 0.3 minutes, about 0.4 minutes, about 0.5 minutes, about
0.6 minutes, about 0.7
minutes, about 0.8 minutes, about 0.9 minutes, about 1 minute, about 1.1
minutes, about 1.2
minutes, about 1.3 minutes, about 1.4 minutes, about 1.5 minutes, about 1.6
minutes, about 1.7
minutes, about 1.8 minutes, about 1.9 minutes, about 2 minutes, about 2.1
minutes, about 2.2
15 minutes, about 2.3 minutes, about 2.4 minutes, about 2.5 minutes, about
2.6 minutes, about 2.7
minutes, about 2.8 minutes, about 2.9 minutes, about 3 minutes, about 3.1
minutes, about 3.2
minutes, about 3.3 minutes, about 3.4 minutes, about 3.5 minutes, about 3.6
minutes, about 3.7
minutes, about 3.8 minutes, about 3.9 minutes, about 4 minutes, about 4.1
minutes, about 4.2
minutes, about 4.3 minutes, about 4.4 minutes, about 4.5 minutes, about 4.6
minutes, about 4.7
20 minutes, about 4.8 minutes, about 4.9 minutes, about 5 minutes, about
5.1 minutes, about 5.2
minutes, about 5.3 minutes, about 5.4 minutes, about 5.5 minutes, about 5.6
minutes, about 5.7
minutes, about 5.8 minutes, about 5.9 minutes, about 6 minutes, about 6.1
minutes, about 6.2
minutes, about 6.3 minutes, about 6.4 minutes, about 6.5 minutes, about 6.6
minutes, about 6.7
minutes, about 6.8 minutes, about 6.9 minutes, about 7 minutes, about 7.1
minutes, about 7.2
25 minutes, about 7.3 minutes, about 7.4 minutes, about 7.5 minutes, about
7.6 minutes, about 7.7
minutes, about 7.8 minutes, about 7.9 minutes, about 8 minutes, about 8.1
minutes, about 8.2
minutes, about 8.3 minutes, about 8.4 minutes, about 8.5 minutes, about 8.6
minutes, about 8.7
minutes, about 8.8 minutes, about 8.9 minutes, about 9 minutes, about 9.1
minutes, about 9.2
minutes, about 9.3 minutes, about 9.4 minutes, about 9.5 minutes, about 9.6
minutes, about 9.7
30 minutes, about 9.8 minutes, about 9.9 minutes, about 10 minutes, about
11 minutes, about 12
minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16
minutes, about 17
minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 22
minutes, about 24
minutes, about 26 minutes, about 28 minutes, about 30 minutes, about 35
minutes, about 40
minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60
minutes. In some
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embodiments, the time interval X comprises time not more than about 60
minutes, not more than
about 55 minutes, not more than about 50 minutes, not more than about 45
minutes, not more than
about 35 minutes, not more than about 30 minutes, not more than about 25
minutes, not more than
about 20 minutes, not more than about 15 minutes, not more than about 10
minutes, not more than
about 5 minutes, not more than about 4 minutes, not more than about 3 minutes,
not more than
about 2 minutes, not more than about 1 minute, not more than about 0.5 minutes
or not more than
about 0.1 minute. In some embodiments, the time interval X comprises time
between about 60
minutes to about 0.1 minute, between about 55 minutes to about 0.5 minute,
between about 50
minutes to about 1 minute, between about 45 minutes to about 2 minute, between
about 40 minutes
to about 3 minutes, between about 35 minutes to about 4 minutes, between about
30 minutes to
about 5 minutes, between about 25 minutes to about 10 minutes or between about
20 minutes to
about 15 minutes.
[0095] In some embodiments, the time interval X comprises time at least about
1 hour, about 2
hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours,
about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours,
about 14 hours,
about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19
hours, about 20 hours,
about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25
hours, about 26 hours,
about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31
hours, about 32 hours,
about 33 hours, about 34 hours, about 35 hours, or about 36 hours. In some
embodiments, the time
interval X comprises time not more than about 36 hours, not more than about 35
hours, not more
than about 34 hours, not more than about 33 hours, not more than about 32
hours, not more than
about 31 hours, not more than about 30 hours, not more than about 29 hours,
not more than about
28 hours, not more than about 27 hours, not more than about 26 hours, not more
than about 25
hours, not more than about 24 hours, not more than about 23 hours, not more
than about 22 hours,
not more than about 21 hours, not more than about 20 hours, not more than
about 19 hours, not
more than about 18 hours, not more than about 17 hours, not more than about 16
hours, not more
than about 15 hours, not more than about 14 hours, not more than about 13
hours, not more than
about 12 hours, not more than about 11 hours, not more than about 10 hours,
not more than about
9 hours, not more than about 8 hours, not more than about 7 hours, not more
than about 6 hours,
.. not more than about 5 hours, not more than about 4 hours, not more than
about 3 hours, not more
than about 2 hours or not more than about 1 hour. In some embodiments, the
time interval X
comprises time between about 36 hours to about 1 hour, between about 35 hours
to about 2 hours,
between about 34 hours to about 3 hours, between about 33 hours to about 4
hours, between about
32 hours to about 5 hours, between about 31 hours to about 6 hours, between
about 30 hours to
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about 7 hours, between about 29 hours to about 8 hours, between about 28 hours
to about 9 hours,
between about 27 hours to about 10 hours, between about 26 hours to about 11
hours, between
about 25 hours to about 12 hours, between about 24 hours to about 13 hours,
between about 23
hours to about 14 hours, between about 22 hours to about 15 hours, between
about 21 hours to
.. about 16 hours, between about 20 hours to about 17 hours or between about
19 hours to about 18
hours.
[0096] In some embodiments, the time interval Y comprises at least about 10
minutes, about 20
minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60
minutes, about 65
minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85
minutes, about 90
.. minutes, about 95 minutes, about 100 minutes, about 150 minutes, about 200
minutes, about 250
minutes, about 300 minutes, about 350 minutes, about 400 minutes, about 450
minutes, about 500
minutes, about 550 minutes, about 600 minutes, about 650 minutes, about 700
minutes, about 750
minutes, about 800 minutes, about 850 minutes, about 900 minutes, about 950
minutes, about 1000
minutes, about 1100 minutes, about 1200 minutes, about 1300 minutes, about
1400 minutes, about
.. 1500 minutes, about 1600 minutes, about 1700 minutes, about 1800 minutes,
about 1900 minutes,
about 2000 minutes, about 2100 minutes, about 2200 minutes, about 2300
minutes, or about 2400
minutes. In some embodiments, the time interval Y comprises time not more than
about 2400
minutes, not more than about 2300 minutes, not more than about 2200 minutes,
not more than
about 2100 minutes, not more than about 2000 minutes, not more than about 1900
minutes, not
more than about 1800 minutes, not more than about 1700 minutes, not more than
about 1600
minutes, not more than about 1500 minutes, not more than about 1400 minutes,
not more than
about 1300 minutes, not more than about 1200 minutes, not more than about 1100
minutes, not
more than about 1000 minutes, not more than about 950 minutes, not more than
about 900 minutes,
not more than about 850 minutes, not more than about 800 minutes, not more
than about 750
minutes, not more than about 700 minutes, not more than about 650 minutes, not
more than about
600 minutes, not more than about 550 minutes, not more than about 500 minutes,
not more than
about 450 minutes, not more than about 400 minutes, not more than about 350
minutes, not more
than about 300 minutes, not more than about 250 minutes, not more than about
200 minutes, not
more than about 150 minutes, not more than about 100 minutes, not more than
about 95 minutes,
not more than about 90 minutes, not more than about 85 minutes, not more than
about 80 minutes,
not more than about 75 minutes, not more than about 70 minutes, not more than
about 65 minutes,
not more than about 60 minutes, not more than about 55 minutes, not more than
about 50 minutes,
not more than about 45 minutes, not more than about 40 minutes, not more than
about 35 minutes,
not more than about 30 minutes, not more than about 25 minutes, not more than
about 20 minutes,
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not more than about 15 minutes or not more than about 10 minutes. In some
embodiments, the time
interval Y comprises time between about 2400 minutes to about 10 minutes,
between about 2300
minutes to about 20 minutes, between about 2200 minutes to about 30 minutes,
between about
2100 minutes to about 40 minutes, between about 2000 minutes to about 50
minutes, between
about 1900 minutes to about 60 minutes, between about 1800 minutes to about 70
minutes, between
about 1700 minutes to about 80 minutes, between about 1600 minutes to about 90
minute, between
about 1500 minutes to about 100 minutes, between about 1400 minutes to about
150 minutes,
between about 1300 minutes to about 200 minutes, between about 1200 minutes to
about 250
minutes, between about 1100 minutes to about 300 minutes, between about 1000
minutes to about
350 minutes, between about 950 minutes to about 400 minutes, between about 900
minutes to
about 450 minutes, between about 850 minutes to about 500 minute, between
about 800 minutes
to about 550 minutes, between about 750 minutes to about 600 minutes or
between about 700
minutes to about 650 minutes.
[0097] In some embodiments, the chamber is pie wedge shaped. In some
embodiments, the
chamber is regular or irregular polygon shaped. In some embodiments, the
chamber is concave
polygon shaped. In some embodiments, the chamber is convex polygon shaped. In
some
embodiments, the chamber is trigon shaped. In some embodiments, the chamber is
quadrilateral
polygon shaped. In some embodiments, the chamber is pentagon shaped. In some
embodiments,
the chamber is hexagon shaped. In some embodiments, the chamber is equilateral
polygon shaped.
In some embodiments, the chamber is equiangular polygon shaped. In some
embodiments, the
chamber is heptagon shaped. In some embodiments, the chamber is octagon
shaped. In some
embodiments, the chamber is nonagon shaped. In some embodiments, the chamber
is decagon
shaped. In some embodiments, the chamber is hendecagon shaped. In some
embodiments, the
chamber is dodecagon shaped. In some embodiments, the chamber is tridecagon
shaped. In some
embodiments, the chamber is tetradecagon shaped. In some embodiments, the
chamber is
pentadecagon shaped. In some embodiments, the chamber is hexadecagon shaped.
In some
embodiments, the chamber is heptadecagon shaped. In some embodiments, the
chamber is
octadecagon shaped. In some embodiments, the chamber is enneadecagon shaped.
In some
embodiments, the chamber is icosagon shaped. In some embodiments, the chamber
is n-gon
shaped. In some embodiments, an edge of the chamber is rounded. In some
embodiments, an edge
of the chamber comprises a baffle.
In some embodiments, the body portion of the chamber comprises at least three
flat polygonal
surfaces. In another embodiment, the body portion of the chamber comprises
one, two, three, four,
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five, six, seven, or eight flat polygonal surfaces. In some embodiments, the
chamber comprises six
flat polygonal surfaces. In some embodiments, one or more of the polygonal
surfaces are rounded.
[0098] In some embodiments, the cross-section of the body portion comprises a
polygonal shape.
In some embodiments, the polygonal shape comprises a trigonal, tetragonal,
pentagonal, or
.. hexagonal shape. In some embodiments, the cross-section of the body portion
comprises a
hexagonal shape. In some embodiments, each chamber of a plurality of chambers,
comprise a
hexagonal shaped cross section. In some embodiments, the plurality of
chambers, each chamber
comprising the hexagonal shaped cross section, comprise a hexagonally packed
arrangement in a
cartridge. In some embodiments, each chamber of the plurality comprising a
hexagonally shaped
cross-section, may be arranged in a honey-comb shaped structure in the
cartridge. In some
embodiments, without being bound to theory, the hexagonal shape may best fill
a plane with equal
size units without leaving out unused space. In some embodiments, hexagonal
packing may
minimize the perimeter for a given area. In some embodiments, hexagonal
packing may minimize
the perimeter for a given area, for example, with 120-degree angles. In some
embodiments, the
hexagonal shape of the chamber ensures less use of raw material.
[0099] In some embodiments, at least a portion of the chamber is circular
shaped. In some
embodiments, at least a portion of the cartridge is regular elliptic shaped.
In some embodiments, a
bottom surface of the chamber is rounded. In some embodiments, a bottom
surface of the chamber
is pointed.
[0100] In some embodiments, the chamber is comprised in a cartridge. In some
embodiments, the
cartridge comprises a plurality of chambers. In further embodiments, the
chambers in the cartridge
have different shapes from each other. In some embodiments, each of the
chambers in the cartridge
have the same or similar shapes. In some instances, the chambers in the
cartridge have mixed
shapes, where at least 25% are of one shape, and at least 25% are another
shape, for example, 35%
.. of the chambers in the cartridge are in the shape of a square, 25% of the
chambers in the cartridge
are in the shape of a wedge and the remaining 40% are in the shape of a
polygon.
[0101] In some embodiments, the first chamber touches at least one side wall
of the second
chamber. In some embodiments, the first chamber does not touch any side wall
of the second
chamber. In some embodiments, the first chamber touches at least one edge of
the second chamber.
.. In some embodiments, the first chamber does not touch any edge of the
second chamber.
[0102] In some embodiments, a material of the chamber comprises a material
that is resistant to,
e.g., cleaning procedures (chemically resistant), extreme temperatures (e.g.,
denaturation of nucleic
acids), extreme pH values (such as those encountered during sanitization of
the reactor with bases
and acids, e.g. with NaOH), mechanical forces (e.g., friction caused by
magnetic particles), and/or
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corrosion. In additional embodiments, the material of the chamber comprises a
material of proper
light permeation (transparent, translucent, or opaque) to a corresponding
purpose. In some
embodiments, the material of the chamber comprises a material of proper gas
permeation to a
corresponding purpose. In further embodiments, the materials of the chamber
should be
5 temperature conductive at working temperatures around 20 C (e.g., W/(mK)
values of at least 10,
preferably at least 15). In some embodiments, the inner surface of the chamber
comprises a surface
material that does not release unwanted compounds that may contaminate the end
product. In
further embodiments, the materials of the chamber and/or the inner surface of
the chamber are PC
(polycarbonate), PP (polypropylene), PAT (polyamide-imide) (e.g. Torlon), PI
(polyimide) (e.g.
10 Tecasint), PPS (polyphenylsulfide) (e.g. Tecatron), PPSU
(polyphenylsulfone) (e.g. Tecason P),
PSU (Polysulfone) (e.g. Tecason S), PEI (polyetherimid) (e.g. Tecapei), glass
(e.g. borosilicate
glass), technical ceramics (e.g. FRIDURITg), Polyaryletherketone (e.g.,
Polyetheretherketon
(PEEK)), thermoplastics (e.g. DuraFormg Pa or DuraFormg GF), all of which
being chemically
resistant, pH resistant, and temperature resistant. In additional embodiments,
the materials of the
15 chamber comprise a material for a single-use including, but not limited
to, polyethylene
terephthalate and other polyethylenes, polyvinyl acetate, polyvinyl chloride.
In some
embodiments, the materials of the chamber comprise a material having
resistance to sterilization
process including steam treatment or ethylene oxide (Et0) exposure/gamma
irradiation even
before adding any reaction-related reagents. In some embodiments, the
materials of the chamber
20 provide protection from light (if needed) for medium contained in the
chamber.
[0103] In some embodiments, the system comprises a plurality of cartridges. In
some
embodiments, the system comprises at least one cartridge, 2 cartridges, 3
cartridges, 4 cartridges,
5 cartridges, 6 cartridges, 7 cartridges, 8 cartridges, 9 cartridges, 10
cartridges, 11 cartridges, 12
cartridges, 13 cartridges, 14 cartridges, 15 cartridges, 16 cartridges, 17
cartridges, 18 cartridges,
25 19 cartridges, 20 cartridges, 21 cartridges, 22 cartridges, 23
cartridges, 24 cartridges, 25 cartridges,
26 cartridges, 27 cartridges, 28 cartridges, 29 cartridges, 30 cartridges, 31
cartridges, 32 cartridges,
33 cartridges, 34 cartridges, 35 cartridges, 36 cartridges, 37 cartridges, 38
cartridges, 39 cartridges,
cartridges, 41 cartridges, 42 cartridges, 43 cartridges, 44 cartridges, 45
cartridges, 46 cartridges,
47 cartridges, 48 cartridges, 49 cartridges, or 50 cartridges. A cartridge of
the plurality of cartridges
30 may comprise at least one chamber. In some embodiments, each cartridge
of the plurality of
cartridges comprises at least one chamber. In some embodiments, a first
cartridge of the plurality
of cartridges comprises the same number of chamber(s) as a second cartridge of
the plurality of
cartridges. In some embodiments, the first cartridge of the plurality of
cartridges comprises a
different number of chamber(s) as the second cartridge of the plurality of
cartridges.
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[0104] In some embodiments, a different reaction occurs in a single and
different cartridge. In an
example, in vitro transcription (IVT) occurs in one cartridge, capping of mRNA
molecules in
another different cartridge, and DNAase treatment in yet another different
cartridge. In some
embodiments, the same reactions occur in all cartridges. In some embodiments,
the same reaction
occurs in a plurality of cartridges. In some embodiments, a first reaction
occurs in a single
compartment and a second rection occurs in a plurality of cartridges. In some
embodiments, n
types of reactions or processes are partitioned across m chambers (which may
be comprised in one
cartridge or spread across a plurality of cartridges, and where n and m are
positive integers and
n < m) such that at least one reaction or process is occurring in each
chamber, but the same type
of reaction or process may occur in more than one chamber, subject to the
constraints of the number
of reactions/processes and chambers.
[0105] In some embodiments, a subset of the plurality of chambers contains,
e.g., medium or
another substance but is not configured to carry out the target process. Such
chambers may
generally be referred to as "blanks." Any proportion of the chambers may be
blanks. In some
embodiments, blanks do not contain any liquid (e.g., medium). In some
embodiments, blanks do
not contain one or more reagents for carrying out the target process (e.g., an
IVT process as
described elsewhere herein).
[0106] In some embodiments, the cartridge comprises a rectangular shape. In
some embodiments,
the cartridge comprises a honeycomb shape. In some embodiments, the cartridge
is circular shaped.
In some embodiments, the cartridge is elliptic shaped. In some embodiments,
the cartridge is crown
shaped. In some embodiments, the cartridge comprises a plurality of rows with
one or more
chambers. In some embodiments, a first row of the plurality of rows has the
same number of
chamber(s) as a second row of the plurality of rows. In some embodiments, the
first row of the
plurality of rows a different number of chamber(s) as a second row of the
plurality of rows.
[0107] Described herein are various embodiments of a compartment for moving a
chamber and/or
cartridge as described herein. Chambers and/or cartridges as disclosed herein
may be comprised in
one or more compartments capable of moving the chambers and/or cartridges. In
some
embodiments, the one or more compartments comprise a carousel. In some
embodiments, the one
or more compartments comprise a conveyor.
[0108] The compartment may be programmed to alter the location of the chambers
and/or
cartridge(s) over time. In some embodiments, the compartment is programmed to
translocate a
chamber and/or cartridge from a first position to a second position then back
to the first position
within a certain time period. The time period may be determined at least in
part by a reaction or
incubation time of a process occurring in the chamber/cartridge; a total
number of cartridges,
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chambers, or compartments in the system; or a combination thereof. In some
embodiments, the
system is configured to carry out a first operation or set of operations at
the first location and a
second operation or set of operations at the second location. In an example
embodiment, a reagent
is added to a chamber at the first location and a synthesized compound is
removed from the
chamber at the second location. In this example embodiment, the compartment is
programmed to
translocate the chamber from the first location to the second location within
a time period Y
corresponding to a reaction time for synthesizing the compound from the
reagent. The
compartment then returns to the first location where the reagent can be added
to the compartment
again, allowing for a virtual "endless" cycle of production of the compound.
In another example
embodiment, a reagent is added to a chamber at a first location. The
compartment then translocates
the chamber to a second location where a second reagent is added to a chamber.
The compartment
then translocates the chamber to a third location where a synthesized compound
is removed.
[0109] The chambers and/or cartridges may be disposed in the carousel. The
carousel may rotate
so as to alter the location of the chambers and/or cartridges over time. The
carousel may be
configured to rotate about an axis in two spatial dimensions. The carousel may
be configured to
rotate at a particular angular speed/velocity. The angular speed/velocity may
be determined by one
or more of an incubation or reaction time of a process in a chamber and/or
cartridge comprised in
the carousel, a desired linear speed/velocity of the chamber and/or cartridge,
a dimension or other
spatial parameter (e.g., length, width, height, volume) of the chamber and/or
cartridge, or any
combination thereof. Alternatively, or additionally, the angular velocity may
be set at a
predetermined rate (e.g., by an operator or by a control system as described
herein).
[0110] In some embodiments, the compartments comprise a conveyor belt.
The conveyor belt
may be configured to move at a particular speed. The speed may be determined
by one or more
one or more of an incubation or reaction time of a process in a chamber and/or
cartridge comprised
in the carousel, a dimension or other spatial parameter (e.g., length, width,
height, volume) of the
chamber and/or cartridge, or any combination thereof. Alternatively, or
additionally, the speed may
be set at a predetermined rate (e.g., by an operator or by a control system as
described herein).
[0111] Devices and systems as described herein may comprise at least one
compartment. In
some embodiments, the system comprises at least two compartments. In some
embodiments, the
system comprises at least three compartments. In some embodiments, the system
comprises at least
four compartments. In some embodiments, the system comprises at least five
compartments. In
some embodiments, the system comprises at least six compartments. In some
embodiments, the
system comprises at least seven compartments. In some embodiments, the system
comprises at
least eight compartments. In some embodiments, the system comprises at least
nine compartments.
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In some embodiments, the system comprises at least ten compartments. In some
embodiments, the
compartments comprise chambers and/or cartridges that are all configured to
carry out the same
process (e.g., synthesis or production of compound, such as a nucleic acid
compound) or subset of
operations comprising a process (e.g., a purification, isolation, and/or
packaging operation which
is part of an overall nucleic acid compound production process) as described
herein. In some
embodiments, one compartment comprises chambers and/or cartridges configured
to perform a
synthesis of a ribonucleic acid molecule, such as by in vitro transcription
(IVT), while a second
compartment comprises chambers and/or cartridges configured to perform
isolation of the
synthesized compound. In such embodiments, the system may be configured to
transport a
substance (e.g., reaction mixture or potion thereof, such as a medium) from
one compartment to
another. The system may transport the substance by any means disclosed
elsewhere herein, such
as by a programmed robotic arm and/or tubing.
[0112] In some embodiments, at least one reagent is added to a chamber in a
cartridge in a
sequential manner. In yet other embodiments, at least one reagent is added to
a portion of the total
number of chambers in a cartridge in a sequential manner. In still other
embodiments, at least one
reagent is added to only one chamber, in two chambers, in three chambers, in
four chambers, in
five chambers or more in a cartridge in a sequential manner. In other
embodiments, an additional
amount of the same reagent is added to a chamber in a cartridge in a
sequential manner. In still
other embodiments, an additional amount of the same reagent is added to a
portion of the total
number of chambers in a cartridge in a sequential manner. In still other
embodiments, an additional
amount of the same reagent is added to only one chamber, in two chambers, in
three chambers, in
four chambers, in five chambers or more in a cartridge in a sequential manner.
[0113] In some embodiments, a medium comprises at least one reagent. In yet
other embodiments,
the medium comprises at least two reagents. In still other embodiments, the
medium comprises
three reagents. In still other embodiments, the medium comprises five or more
reagents. In some
embodiments, the medium with at least one reagent is loaded to a chamber
before the next chamber
is loaded in a sequential manner. In some embodiments, the medium with two
reagents is loaded
to a chamber before the next chamber is loaded in a sequential manner. In some
embodiments, the
medium with five or more reagents is loaded to a chamber before the next
chamber is loaded in a
sequential manner. In some embodiments, the reagents are pre-mixed in a
separate premixing
chamber and the premixed reagents are loaded to a chamber for reaction.
[0114] In some embodiments, at least one reagent is reloaded to a chamber in a
cartridge after the
last chamber in the cartridge is loaded with at least one reagent. In some
embodiments, at least one
reagent is reloaded to a chamber in a cartridge in a sequential manner after
the last chamber in the
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cartridge is loaded with at least one reagent. In yet other embodiments, at
least one reagent is
reloaded to a portion of the total number of chambers in a cartridge in a
sequential manner after
the last chamber in the cartridge is loaded with at least one reagent. In
still other embodiments, at
least one reagent is reloaded in only one chamber, in two chambers, in three
chambers, in four
chambers, in five chambers or more in a cartridge in a sequential manner after
the last chamber in
the cartridge is loaded with at least one reagent. In some embodiments, at
least one reagent is
reloaded to a chamber in a cartridge in a sequential manner after the last
chamber in the cartridge
is loaded with at least one reagent, and the reagent is the same as the
reagent previously loaded. In
yet other embodiments, at least one reagent is reloaded to a portion of the
total number of chambers
in a cartridge in a sequential manner after the last chamber in the cartridge
is loaded with at least
one reagent, and the reagent is the same as the reagent previously loaded. In
still other
embodiments, at least one reagent is reloaded in only one chamber, in two
chambers, in three
chambers, in four chambers, in five chambers or more in a cartridge in a
sequential manner after
the last chamber in the cartridge is loaded with at least one reagent, and the
reagent is the same as
the reagent previously loaded. In some embodiments, at least one reagent is
reloaded to a chamber
in a cartridge in a sequential manner after the last chamber in the cartridge
is loaded with at least
one reagent, and the reagent is different from the reagent previously loaded.
In yet other
embodiments, at least one reagent is reloaded to a portion of the total number
of chambers in a
cartridge in a sequential manner after the last chamber in the cartridge is
loaded with at least one
reagent, and the reagent is different from the reagent previously loaded. In
still other embodiments,
at least one reagent is reloaded only one chamber, in two chambers, in three
chambers, in four
chambers, in five chambers or more in a cartridge in a sequential manner after
the last chamber in
the cartridge is loaded with at least one reagent, and the reagent is
different from the reagent
previously loaded.
[0115] In some embodiments, the chambers of the cartridge can be reused
multiple times. In further
embodiments, the chambers are rinsed/washed with at least one solution between
each reaction
cycle. In additional embodiments, the solution comprises deionized water,
purified water, a
buffered solution, a cleaning solution, or a combination thereof
[0116] In some embodiments, a chamber in a cartridge can be drained in a
sequential manner
before the last chamber in the same cartridge is loaded with at least one
reagent. In yet other
embodiments, a portion of the total number of chambers in a cartridge can be
drained in a sequential
manner before the last chamber in the same cartridge is loaded with at least
one reagent. In still
other embodiments, only one chamber, two chambers, three chambers, four
chambers, five
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chambers or more in a cartridge can be drained in a sequential manner before
the last chamber in
the same cartridge is loaded with at least one reagent.
[0117] In some embodiments, the first chamber in the cartridge can be reloaded
with at least one
reagent before the chamber is drained, and the at least one reagent is the
same as the previously
5 loaded reagent. In another embodiment, the first chamber in the cartridge
can be reloaded with at
least one reagent before the chamber is drained, and the at least one reagent
is not the same as the
previously loaded reagent.
[0118] In some embodiments, the contents of the chambers are agitated (e.g.,
stirred), so as to
facilitate mixing of the contents of the chambers. In some embodiments, the
contents of the
10 chamber are mixed by vibrating or shaking the chamber. In yet other
embodiments, each of the
chambers are simultaneously mixed. In other embodiments, the chambers in a
cartridge are mixed
independently of each other. In yet other embodiments, an impeller mixes the
contents of the
chamber. In still other embodiments, the contents of the chamber are mixed by
repeated pipetting
of the contents.
15 [0119] In some embodiments, the chamber comprises at least one baffle,
which mixes the contents
of the chamber. In some embodiments, the chamber comprises an agitation
device. In some
embodiments, the agitation device comprises an impeller blade, a bead, a
stirrer, a magnetic particle
(e.g., bead), or a magnetic bar. In additional embodiments, the chamber
comprises a mixer, the
shaft of which can be made to rotate by a drive, as a result of which a
stirring member connected
20 rigidly to the mixer shaft is likewise made to rotate, thus mixing the
substances present in the
chamber. In some embodiments, the chamber comprises two or more stirring
members, mostly
spaced axially apart, to be arranged on and connected to the mixer shaft. The
stirring member or
stirring members may also be integral with the mixer shaft. In some
embodiments, the mixer can
be made to rotate by a magnetic drive. In further embodiment, the chamber is
coupled to a control
25 device or programmed to control in real-time a rate of agitation of the
medium. The purposes of
agitation or mixing include, but are not limited to, maintaining the
homogeneity of the reaction
mixture or medium and prevention of suspended particles or cells.
[0120] Described herein are various embodiments, of a hexagonally packed
cartridge, the cartridge
comprising: a plurality of hexagonal chambers, or tubes, the hexagonal
chambers, or tubes
30 configured for in vitro transcription (IVT) (e.g., to produce an mRNA
molecule) and down-stream
processing (DSP), and wherein cartridge is configured for each of the
hexagonal chambers of the
plurality to be arranged in a hexagonally-packed configuration. In some
embodiments, the DSP
comprises post-transcriptional capping or purification of the mRNA molecule.
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[0121] Described herein are various embodiments, of a rectangularly packed
cartridge, the
cartridge comprising: a plurality of hexagonal chambers (e.g., chambers which
comprise a
hexagonal cross-section) or tubes, the hexagonal chambers or tubes configured
for in vitro
transcription (IVT) (e.g., to produce an mRNA molecule) and down-stream
processing (DSP), and
.. wherein the cartridge is configured for each of the hexagonal chambers of
the plurality to be
arranged in a rectangularly-packed configuration. In some embodiments, the DSP
comprises post-
transcriptional capping or purification of the mRNA molecule.
Lid
.. [0122] In some embodiments, the chamber comprises a cover or lid, to
prevent unwanted
components from entering the chamber (for example, RNases, microbial
contamination or other
degrading compounds or organisms) and from shielding the content of the
chamber from the outer
environment. In some embodiments, the chamber comprises the lid to limit
exchange with the
environment. In some embodiment, the cartridge comprises a lid. In some
embodiments, the lid of
the cartridge is removable. In some embodiments, the lid of the cartridge is
not removable.
[0123] In some embodiments, the lid can prevent excessive water evaporation
and loss of other
critical volatile components. In some embodiments, the lid can prevent
oxidation of the reagents
or any components. In some embodiments, the lid can provide with protection
from light (if
needed). In some embodiments, the lid prevents contamination from any other
potential chemical
compound.
[0124] In some embodiments, the lid of the chamber is removable. In some
embodiments, the lid
of the chamber is not removable. In some embodiments, the lid of the chamber
comprises at least
one opening for filling, draining and sampling. In some embodiments, the at
least one opening is
positioned on the top of the lid. In some embodiments, the lid of the chamber
covers the first and
possibly also the second opening.
[0125] In additional embodiments, the sample taken from the chamber through
the at least one
opening in the lid is transferred to, for example but not limited to, laser-
based analyzers such as a
flow cytometry and cell sorting devices.
[0126] In some embodiments, the cartridge is programmed to shake to mix at
least one reagent.
In some embodiments, the all chambers of the cartridge are configured to be
shaken by an d motion
(e.g., by an orbital shaker). In some embodiments, a portion of the chambers
of the cartridge is
configured to be shaken by an oscillating motion. In further embodiment, the
cartridge is coupled
to a control device or programmed to control in real-time a shaking motion.
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[0127] In some embodiments, the chambers are configured to move on a conveyor
belt powered
by one or more powered pulleys. In some embodiments, the chamber is removable
from the
conveyor. In some embodiments, the chamber is not removable from the conveyor.
In some
embodiments, the chambers could be linked to the conveyor system using a
rotation link.
[0128] In some embodiments, the chambers are configured to move within a
carousel in which the
chambers are disposed. In some embodiments, the chamber is removable from the
carousel. In
some embodiments, the chamber is not removeable from the carousel.
[0129] In some embodiments, the chamber comprises a lid. In some embodiments,
the lid may be
removable. In some embodiments, the lid may be configured to prevent unwanted
components
from entering the chamber. In some embodiments, the lid may be configured to
shield the content
of the chamber from the outer environment. In an embodiment, the lid may be
configured to prevent
evaporation of the contents of the chamber. In some embodiments, the open end
of the chamber
may be configured to receive the removable lid for at least partially closing
the open end. In some
embodiments, the lid comprises a round, square, rectangle, polygon, ellipsoid,
triangle, pentagon,
heptagon, octagon, nonagon, decagon, hendecagon, dodecagon, tridecagon,
tetradecagon,
pentadecagon, hexadecagon, heptadecagon, octadecagon, enneadecagon, icosagon,
n-gon shaped,
elliptic, or hexagon shape.
[0130] In some embodiments, the system comprises a chamber and a removable lid
for at least
partially closing the open end of the chamber. In some embodiments, the
removable lid may be
configured to ensure shielding the content of the chamber from the environment
and also prevents
evaporation.
[0131] In some embodiments, the lid comprises a push-on lid. In another
embodiment, the lid
comprises, for example, a screw lid, a flip top, a crown cap, a snap-on lid, a
friction fit lid, a lug
cap, a dome cap, a pail lid, a ribbed closure, a smooth closure, a tub lid or
combinations thereof.
.. In some embodiments, the lid is bonded to at least a portion of the chamber
or a cartridge
comprising the chamber. In some embodiments, the lid is adhered to at least a
portion of the
chamber or a cartridge comprising the chamber. In some embodiments, the lid is
glued to at least
a portion of the chamber or a cartridge comprising the chamber.
[0132] In some embodiments, the lid comprises an opening. In some embodiments,
the lid
comprises a central opening. In some embodiments, the opening may be
configured for access to
a handling mechanism, the handling mechanism configured to add or remove
components and/or
liquids in one or more chambers.
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[0133] In some embodiments, the lid comprises a puncturable membrane. In some
embodiments,
the puncturable membrane is an elastic membrane. In some embodiments, the
assembly comprises
a valve or inlet.
Identification (ID) mechanism
[0134] In some embodiments, the chamber may comprise an identification (ID)
mechanism. In
some embodiments, the ID mechanism may comprise an RFID tag, a smart label, a
reader for
reading an RFID tag or smart label, or a combination thereof In some
embodiments, the Smart
labels may comprise QR codes, bar codes, or a combination thereof. In some
embodiments,
processing steps of the chamber may be determined by the ID of the chamber. In
some
embodiments, the ID of the chamber may be transmitted to a processor, which
based on this
information determines the processing step of the chambers and transmits a
signal to a processing
mechanism to execute the step. In an embodiment, the cartridge may comprise
one or more ID
mechanisms. In some embodiments, the cartridge may comprise an RFID tag.
Aperture
In some embodiments, the cartridge comprises at least one aperture. In some
embodiments, the
aperture is configured to engage with a gripping mechanism. In some
embodiments, a robotic arm
or handling device comprises the gripping mechanism. In some embodiments, the
cartridge
comprises at least two apertures. In some embodiments, the apertures are
positioned at the upper
surface of the cartridge.
Robotic Arm and/or Handling Device
[0135] In some embodiments described herein, the robotic arm is programmed to
operate by the at
least one computer processor. In some embodiments, the robotic arm is a static
arm under the
control of at least one computer processor. In some embodiments, the robotic
arm is movable and
under the control of at least one computer processor. In still other
embodiments, the robotic arm is
capable of moving in at least an X-axis of direction relative to the
cartridge. In other embodiments,
the robotic arm is capable of moving in at least a Y-axis of direction
relative to the cartridge. In
other embodiments, the robotic arm is capable of moving in at least a Z-axis
of direction relative
to the cartridge. In one embodiment, the robotic arm is capable of moving in
both an X-axis and a
Y-axis relative to the cartridge. In other embodiments, the robotic arm is
capable of moving in at
least two directions consisting of an X-axis, Y-axis and Z-axis relative to
the cartridge.
[0136] In some embodiments, the system comprises at least one robotic arm. In
some
embodiments, the system comprises one robotic arm. In some embodiments, the
system comprises
two robotic arms. In some embodiments, the system comprises three robotic
arms. In some
embodiments, the system comprises four robotic arms. In some embodiments, the
system
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comprises five robotic arm. In some embodiments, the system comprises six
robotic arms. In some
embodiments, the system comprises seven robotic arms. In some embodiments, the
system
comprises eight robotic arms. In some embodiments, the system comprises nine
robotic arms. In
some embodiments, the system comprises ten robotic arms. In some embodiments,
the system
comprises eleven robotic arms. In some embodiments, the system comprises
twelve robotic arms.
[0137] In some embodiments, the robotic arm is human finger shaped. In some
embodiments, the
robotic arm is human hand shaped. In some embodiments, the robotic arm is
human arm shaped.
In some embodiments, the robotic arm is one arm. In some embodiments, the
robotic arm is one
hand. In some embodiments, the robotic arm is five fingers. In some
embodiments, the robotic arm
is two arms. In some embodiments, the robotic arm is two hands. In some
embodiments, the robotic
arm is ten fingers.
[0138] In some embodiments, the robotic arm is positioned in the vicinity of
the cartridge in a
manner that each chamber can be reached by the robot and the robot arm is
covering all the
chambers. In some embodiments, the chambers are movable and may be moved
closer to the robot
arm to dispense or remove medium and/or reagent from a chamber (e.g., by a
carousel or conveyor
belt). In some embodiments, a plurality of chambers is positioned to surround
a robotic arm. In
some embodiment, the robotic arm is located above the chambers or can be
installed next to the
cartridge.
[0139] In some embodiments, the at least one robotic arm is removable from the
system. In another
embodiments, the at least one robotic arm is not removable from the system.
[0140] In some embodiments, the at least one robotic arm loads the at least
one reagent to the
chambers. In some embodiments, the at least one robotic arm removes the
compound from the
chamber.
[0141] In some embodiments, the system comprises one or more robotic arms. In
some instances,
such as when a plurality of robotic arms is present, different reagents can be
provided by different
robots. In some instances, when more than one robotic arm is present,
evacuation of the chamber
content can be performed by a separate robotic arm. In some embodiments, the
evacuation of the
content of the chamber is performed by the same robotic arm. In some
embodiments, the number
of robotic arms is determined at least in part based on a time interval
between filling a first chamber
and a second chamber (sometimes referred to herein as a time interval X), a
time interval between
adding a medium or portion thereof to a chamber and removing a synthesized
compound from the
chamber (sometimes referred to herein as a time interval Y), a number of
chambers, a number of
cartridges, or any combination thereof In an example embodiment, a robotic arm
is programmed
to sequentially add to a plurality of chambers a reagent which participates in
the synthesis of a
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compound. The robotic arm is further programmed to remove the synthesized
compound from the
plurality of chambers after a time interval Y, where Y is a reaction or
incubation time for producing
a compound with the added reagent. The synthesized compound may be comprised
in a medium
which is removed from the chamber by the robotic arm. In another example
embodiment, a robotic
5 arm is programmed to sequentially add to a plurality of chambers a
reagent which participates in
the synthesis of a compound. A second robotic arm is programmed to remove the
synthesized
compound from the plurality of chambers after a time interval Y, where Y is a
reaction or
incubation time for producing a compound with the added reagent. The
synthesized compound
may be comprised in a medium which is removed from the chamber by the second
robotic arm.
10 [0142] In some embodiments, the at least one robotic arm is equipped
with pipettes, micropipettes,
needles or tips for delivering the reagents to the chambers. In some
embodiment, the pipettes,
micropipettes, needles or tips is removable and/or single-use. In some
embodiment, the pipettes,
micropipettes, needles or tips is not removable.
[0143] In some embodiments, the at least one robotic arm is programmed to
transfer the at least
15 one reagent to and from the chambers.
[0144] In some embodiments, the at least one robotic arm is programmed to add
the reagents to
the chamber during the reaction, if necessary.
[0145] In some embodiments, the programming of the at least one robotic arm is
based at least in
part on the number of chamber and/or cartridges, a time interval between
adding or removing a
20 substance form subsequent chambers, a reaction or incubation time, an
overall processing time, or
any combination thereof.
Separation and/or Purification Device
[0146] The present disclosure provides various embodiments of a device for
separating and/or
purifying a compound, the compound in accordance with some embodiments. In
some
25 embodiments, the device for separating and/or purifying a compound
comprise a chamber, a
cartridge, a lid, a sensor, or any combination thereof, as described elsewhere
herein with respect to
reactor systems for, e.g., production and/or synthesis of the compound. In
some embodiments, the
device for separating and/or purifying the compound is the same as the device
for producing and/or
synthesizing the compound or is comprised in a part of the device or system
for producing and/or
30 synthesizing the compound. In some embodiments, the device for
separating and/or purifying the
compound is separate from the device for producing and/or synthesizing the
compound. In such
embodiments, the compound is transferred from the synthesis device to the
purification/separation
device by manual intervention (e.g., of a human operator) or automatically,
such as by a robotic
arm or other handling device as described elsewhere herein.
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[0147] In some embodiments, the device for separating and/or purifying a
compound comprises
a carousel. FIG. 1 depicts a non-limiting example a carousel 100. In some
embodiments, the device
for separating and/or purifying a compound may be automated. In some
embodiments, the device
for separating and/or purifying a compound may comprise a sample plate. In
some embodiments,
the sample plate may comprise sample holders. In some embodiments, the sample
holders are
configured to hold sample containers comprising the compound in a liquid
medium in a certain
position. In some embodiments, a magnet unit may be positioned at each sample
holder. In some
embodiments, magnetic particles may be introduced into the samples and mixed
with the
compound. In some embodiments, magnetic particle-bound compound may be
subjected to
washing and elution steps, inside the device. In some embodiments, the
compound is referred to as
the compound of interest. In the same or another embodiment, the carousel 100
depicted in FIG.
1 is used for production of the compound.
[0148] In one aspect, robotic arms, injectors, and/or pumps are used for
dispensing and removing
components and liquids in the sample containers positioned within the device
for separating and/or
purifying a compound.
[0149] In some embodiments, the device for separating and/or purifying a
compound comprises:
a sample plate, the sample plate comprising a base portion and one or more
sample holders
provided on the base portion for receiving a sample container, the sample
container configured to
hold a liquid sample comprising the compound and magnetic particles; and a
magnet unit
positioned at each sample holder, the magnet unit configured to capture the
magnetic particles or
introduce a movement of the magnetic particles, and wherein the sample holders
are configured to
perform a mechanical motion, such that the magnetic particles are mixed with
the liquid.
[0150] System Controller (Computer Processor)
[0151] The present disclosure provides computer control systems that are
programmed to
implement methods of the disclosure. FIG. 10 shows a computer system 101 that
is programmed
or otherwise configured to control at least one robotic arm and the system.
The computer system
101 can regulate various aspects of the present disclosure, such as, for
example, performance of at
least one robotic arm. The computer system 101 can be an electronic device of
a user or a computer
system that is remotely located with respect to the electronic device. The
electronic device can be
a mobile electronic device.
[0152] The computer system 101 includes a central processing unit (CPU, also
"processor" and
"computer processor" herein) 105, which can be a single core or multi core
processor, or a plurality
of processors for parallel processing. The computer system 101 also includes
memory or memory
location 110 (e.g., random-access memory, read-only memory, flash memory),
electronic storage
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unit 115 (e.g., hard disk), communication interface 120 (e.g., network
adapter) for communicating
with one or more other systems, and peripheral devices 125, such as cache,
other memory, data
storage and/or electronic display adapters. The memory 110, storage unit 115,
interface 120 and
peripheral devices 125 are in communication with the CPU 105 through a
communication bus
(solid lines), such as a motherboard. The storage unit 115 can be a data
storage unit (or data
repository) for storing data. The computer system 101 can be operatively
coupled to a computer
network ("network") 130 with the aid of the communication interface 120. The
network 130 can
be the Internet, an internet and/or extranet, or an intranet and/or extranet
that is in communication
with the Internet. The network 130 in some cases is a telecommunication and/or
data network.
The network 130 can include one or more computer servers, which can enable
distributed
computing, such as cloud computing. The network 130, in some cases with the
aid of the computer
system 101, can implement a peer-to-peer network, which may enable devices
coupled to the
computer system 101 to behave as a client or a server.
[0153] The CPU 105 can execute a sequence of machine-readable instructions,
which can be
embodied in a program or software. The instructions may be stored in a memory
location, such as
the memory 110. The instructions can be directed to the CPU 105, which can
subsequently
program or otherwise configure the CPU 105 to implement methods of the present
disclosure.
Examples of operations performed by the CPU 105 can include fetch, decode,
execute, and
writeback.
[0154] The CPU 105 can be part of a circuit, such as an integrated circuit.
One or more other
components of the system 101 can be included in the circuit. In some cases,
the circuit is an
application specific integrated circuit (ASIC).
[0155] The storage unit 115 can store files, such as drivers, libraries and
saved programs. The
storage unit 115 can store user data, e.g., user preferences and user
programs. The computer system
101 in some cases can include one or more additional data storage units that
are external to the
computer system 101, such as located on a remote server that is in
communication with the
computer system 101 through an intranet or the Internet.
[0156] The computer system 101 can communicate with one or more remote
computer systems
through the network 130. For instance, the computer system 1101 can
communicate with a remote
computer system of a user. Examples of remote computer systems include
personal computers
(e.g., portable PC), slate or tablet PC's (e.g., Apple iPad, Samsung Galaxy
Tab), telephones,
Smart phones (e.g., Apple iPhone, Android-enabled device, Blackberry ), or
personal digital
assistants. The user can access the computer system 101 via the network 130.
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[0157] Methods as described herein can be implemented by way of machine (e.g.,
computer
processor) executable code stored on an electronic storage location of the
computer system 101,
such as, for example, on the memory 110 or electronic storage unit 115. The
machine executable
or machine-readable code can be provided in the form of software. During use,
the code can be
executed by the processor 105. In some cases, the code can be retrieved from
the storage unit 115
and stored on the memory 110 for ready access by the processor 105. In some
situations, the
electronic storage unit 115 can be precluded, and machine-executable
instructions are stored on
memory 110.
[0158] The code can be pre-compiled and configured for use with a machine
having a processer
adapted to execute the code or can be compiled during runtime. The code can be
supplied in a
programming language that can be selected to enable the code to execute in a
pre-compiled or as-
compiled fashion.
[0159] Aspects of the systems and methods provided herein, such as the
computer system 101, can
be embodied in programming. Various aspects of the technology may be thought
of as "products"
or "articles of manufacture" typically in the form of machine (or processor)
executable code and/or
associated data that is carried on or embodied in a type of machine readable
medium. Machine-
executable code can be stored on an electronic storage unit, such as memory
(e.g., read-only
memory, random-access memory, flash memory) or a hard disk. "Storage" type
media can include
any or all of the tangible memory of the computers, processors or the like, or
associated modules
thereof, such as various semiconductor memories, tape drives, disk drives and
the like, which may
provide non-transitory storage at any time for the software programming. All
or portions of the
software may at times be communicated through the Internet or various other
telecommunication
networks. Such communications, for example, may enable loading of the software
from one
computer or processor into another, for example, from a management server or
host computer into
the computer platform of an application server. Thus, another type of media
that may bear the
software elements includes optical, electrical and electromagnetic waves, such
as used across
physical interfaces between local devices, through wired and optical landline
networks and over
various air-links. The physical elements that carry such waves, such as wired
or wireless links,
optical links or the like, also may be considered as media bearing the
software. As used herein,
unless restricted to non-transitory, tangible "storage" media, terms such as
computer or machine
"readable medium" refer to any medium that participates in providing
instructions to a processor
for execution.
[0160] Hence, a machine readable medium, such as computer-executable code, may
take many
forms, including but not limited to, a tangible storage medium, a carrier wave
medium or physical
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transmission medium. Non-volatile storage media include, for example, optical
or magnetic disks,
such as any of the storage devices in any computer(s) or the like, such as may
be used to implement
the databases, etc. shown in the drawings. Volatile storage media include
dynamic memory, such
as main memory of such a computer platform. Tangible transmission media
include coaxial cables;
copper wire and fiber optics, including the wires that comprise a bus within a
computer system.
Carrier-wave transmission media may take the form of electric or
electromagnetic signals, or
acoustic or light waves such as those generated during radio frequency (RF)
and infrared (IR) data
communications. Common forms of computer-readable media therefore include for
example: a
floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic
medium, a CD-ROM,
DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other
physical storage
medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM,
any
other memory chip or cartridge, a carrier wave transporting data or
instructions, cables or links
transporting such a carrier wave, or any other medium from which a computer
may read
programming code and/or data. Many of these forms of computer readable media
may be involved
in carrying one or more sequences of one or more instructions to a processor
for execution.
[0161] The computer system 101 can include or be in communication with an
electronic display
135 that comprises a user interface (UI) 140 for providing. Examples of UI' s
include, without
limitation, a graphical user interface (GUI) and web-based user interface.
[0162] Methods and systems of the present disclosure can be implemented by way
of one or more
algorithms. An algorithm can be implemented by way of software upon execution
by the central
processing unit 105.
Sensors
[0163] In some embodiments described herein, the system comprises one or more
sensors or
probes for monitoring one or more operational parameters in real-time
including, but not limited
to, a liquid level sensor, a thermometer, a pH probe, an oxygen probe, a
carbon dioxide probe, a
rate of agitation sensor, combinations thereof, and the like. These sensors
can detect one or more
operational parameters and/or monitor physical properties and chemical
compositions,
combinations thereof, and the like of the medium or the chamber headspace.
[0164] In some embodiments, the chamber is configured to detect a
contamination of the at least
one reagent. In some embodiments, the chamber is configured to detect a
contamination of more
than one reagent.
[0165] In some embodiments, the chamber is configured to monitor and/or
regulate a pH value of
a medium comprising the at least one reagent. In further embodiments, the
chamber comprises a
pH probe to monitor a pH value of a medium real-time.
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[0166] In some embodiments, the chamber is configured to monitor and/or
regulate a temperature
of a medium comprising the at least one reagent or an atmosphere with the
chamber. In further
embodiments, the chamber comprises a thermometer. In additional embodiments,
the chamber
comprises a thermal probe to monitor a temperature of a medium in real-time.
5 [0167] In some embodiments, the chamber is configured to monitor and/or
regulate a salt
concentration of a medium comprising the at least one reagent.
[0168] In some embodiments, the chamber is configured to monitor and/or
regulate osmolarity of
a medium comprising the at least one reagent. In some embodiments, the chamber
comprises a
device for measuring osmolarity of a medium in real-time.
10 [0169] In some embodiments, the chamber is configured to monitor and/or
regulate conductivity
of a medium comprising the at least one reagent. In some embodiments, the
chamber comprises an
electrical conductivity meter to monitor conductivity of a medium in real-
time.
[0170] In some embodiments, the chamber is configured to monitor and/or
regulate turbidity of
the medium comprising the at least one reagent. In further embodiments, the
chamber comprises a
15 turbidity tube to monitor turbidity in real-time.
[0171] In some embodiments, the chamber is configured to monitor and/or
regulate a rate of
agitation of the medium comprising the at least one reagent. In some
embodiments, the chamber
comprises an agitation sensor to monitor a rate of agitation of the medium in
real-time.
[0172] In some embodiments, the chamber is configured to monitor and/or
regulate volume of the
20 medium comprising the at least one reagent. In some embodiments, the
chamber comprises a liquid
lever sensor to monitor volume of the medium in real-time.
[0173] In some embodiments, the chamber is configured to monitor and/or
regulate humidity of
the atmosphere within the chamber. In additional embodiments, the chamber
comprises a
hygrometer to monitor humidity of the atmosphere within the chamber.
25 [0174] In some embodiments, the chamber is configured to monitor and/or
regulate 02
concentration of the atmosphere within the chamber. In some embodiments, the
chamber
comprises an oxygen probe to monitor oxygen concentration of the atmosphere in
real-time.
[0175] In some embodiments, the chamber is configured to monitor and/or
regulate CO2
concentration of the atmosphere within the chamber. In some embodiments, the
chamber
30 comprises a carbon dioxide probe to monitor CO2 concentration of the
atmosphere within the
chamber in real-time.
[0176] In some embodiments, the chamber is configured to monitor and/or
regulate gas
composition of the atmosphere within the chamber. In some embodiments, the
chamber is
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configured to monitor and/or regulate gas composition of dissolved gases in
the medium. In some
embodiments, the dissolved gases comprise oxygen, carbon dioxide, or both.
[0177] In some embodiments, the chamber is configured to monitor and/or
regulate a concentration
of a molecule comprised in the medium utilizing a spectroscopic device. In
some embodiments,
the spectroscopy device comprises Infrared spectrometer, Raman spectrometer or
UV
spectrometer.
[0178] In some embodiments, the chamber is configured to monitor and/or
regulate a viable cell
density based on a cell concentration measurement. In some embodiments, the
chamber comprises
a capacitance probe to monitor the cell concentration within the chamber.
[0179] In some instances, if necessary, an aliquot of the content in the
chamber is taken/transferred
from the chamber during the reaction so that physical properties and/or
chemical compositions of
the content can be monitored in real-time. In some embodiments, the aliquot of
the content in the
chamber is taken/transferred by at least one robotic arm. In further
embodiments, the chambers are
connected to an autosampler that takes samples and routes them to any
intermediate sample
processing machines and/or automated analyzers.
[0180] In some embodiments, the chamber comprises an open end configured to
receive at least
one reagent, wherein at least part of the body portion comprises a flat
polygonal surface configured
to allow positioning and/or supporting a sensor in the chamber. In some
embodiments, the chamber
comprises a sensor and/or a probe. In some embodiments, sensors and/or probes
contribute to the
monitoring of processes that take place within the chamber. In some
embodiments, the sensor
comprises: a liquid level sensor; a rate of agitation sensor; a sensor or
probe that measures the pH,
the temperature, the pressure, the flow velocity, or the oxygen, carbon
dioxide, magnesium, or salt
levels of the components contained in the chamber; or a combination thereof.
In some
embodiments, the sensor may comprise a longitudinal insert, located inside the
chamber.
[0181] In some embodiments, the chamber is configured for single use. In some
embodiments, the
chamber is configured for multiple uses.
Additional Components
[0182] In some embodiments disclosed herein, a chamber, a storage vessel, and
a robotic arm are
configured respectively to transmit data.
[0183] In some embodiments, the system comprises at least one intermediate
storage vessel. In
some embodiments, the intermediate storage vessel is configured to store the
compound. In some
embodiments, the intermediate storage vessel is not in fluid communication
with the chamber. In
some embodiments, the storage vessel is configured to detect a contamination
of the compound.
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[0184] In some embodiments, the intermediate storage vessel is configured to
being coupled to
one or more sensors or probes for monitoring one or more physical properties
and chemical
compositions, combinations thereof, and the like of the compound. In some
embodiments, the
intermediate storage vessel contains means to perform certain chemical,
physical or mechanical
treatment of the compound and/or the undesired residuals ¨ for example,
binding of the product,
partial elimination of residuals, or adjustment of pH value.
[0185] In some embodiments, the system comprises a purification system. In
further embodiments,
the purification system comprises an affinity purification system, an ion
exchange system, a
selective precipitation system, a chromatography system, a tangential flow
filtration device or a
dead end filtration device.
[0186] In some embodiments, the system comprises separate units for the
delivery of reagents and
the evacuation of waste, as well as for the collection of the biochemical
compound. Each unit is
thus designed to allow the provision of filtered, sterile air to be circulated
within the units. Air
filtering means comprises a HVAC system with HEPA filters. In some
embodiments, the system
comprises a HVAC system comprising a series of HEPA filters to protect the
compound within the
chamber.
[0187] In some embodiments, the compound is removed from the chamber by a
pressurized
system. In some embodiments, the pressurized system comprises a negative
pressure. In some
embodiments, the pressurized system comprises a pump configured to remove a
medium
comprising the compound. In some embodiments, the pump is in fluid
communication with a
chamber. In some embodiments, the pump is not in fluid communication with a
chamber. In
additional embodiments, the pump comprises a vacuum pump, a peristaltic pump,
a centrifugal
pump or combination thereof. In some embodiments, the compound is removed from
the chamber
by at least one robotic arm which is equipped with pipettes, micropipettes,
needles or tips.
[0188] In some embodiments, the system comprises a modular unit. In some
embodiments, the
modular unit comprises a docking station configured to receive a receiving
container, a cartridge
or a conveyor.
[0189] In some embodiments, the modular unit comprises at least one cartridge.
In some
embodiments, the modular unit is movable. In some embodiments, the modular
unit is stackable.
In some embodiments, the modular unit comprises a plurality of receiving
containers. In some
embodiments, the receiving container is configured to contain at least one
reagent.
[0190] In some embodiments, different modular units are assembled or
constructed on site rapidly,
and potentially disassembled with similar rapidity. In some embodiments, if
necessary, the modular
units and/or the cartridges can be added for a particular need or process
setting. In some
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embodiment, the modular unit is configured to be coupled to a heat transfer
device. In some
embodiments, the modular unit comprises an agitation device. In some
embodiments, the modular
unit comprises one or more robotic arms. In some embodiments, the modular unit
comprises one
or more pumps. In some embodiments, the modular unit comprises a purification
system. In some
embodiments, the modular unit comprises an air filtration system. In some
embodiments, the
modular unit comprises a controller system.
[0191] In further embodiments, the two separate cartridges can accommodate two
separate steps
of the reaction so that the first step of the reaction occurs in a first
cartridge and the second step of
the reaction occurs in a second cartridge. In some embodiments, the reaction
conditions and/or
reaction parameters of two separate steps of the reaction are separately
controlled by the computer
processor.
[0192] In some embodiments, the plurality of chambers is disposed on the
conveyor. In some
embodiments, the modular unit comprises at least one conveyor. In some
embodiments, at least
one conveyor is stackable.
[0193] In some embodiments, the system comprises a waste collecting vessel,
which facilitates
draining and replacement in a controlled manner. In some embodiments, the
waste collecting vessel
may be connected to the system with a valve that is turned on to remove waste
from the system
when desired.
Methods
[0194] Provided herein, in one embodiment, is a method of producing a
compound, comprising:
(a) providing a plurality of chambers, and a chamber of the plurality of
chambers comprises a
medium comprising at least one reagent; and (b) using at least one computer
processor to generate
instructions to: (i) fill a chamber with the medium; (ii) repeat (i) until at
least a portion of the
plurality of chambers is filled with the medium; (iii) synthesize the compound
in a chamber that is
filled with the medium and remove the compound from the chamber that is filled
with the medium
after a time interval Y; (iv) rinse/wash the chamber and (v) continuously re-
load, synthesize, and
remove the compound.
[0195] In some embodiments, the method comprises removing the compound from
the chamber.
In some embodiments, the method comprises removing the medium through a filter
so that the
compound is retained in the chamber.
[0196] In some embodiments of the present disclosure, the method comprises
providing a robotic
arm coupled to the at least one computer processor and configured to receive
the instructions in
(b).
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[0197] In some embodiments, the method comprises filling a subsequent chamber
after a time
interval X. In some embodiments, the time interval Y is at least the time
interval X times the
number of the plurality of chambers.
[0198] In some embodiments are described methods comprising the at least one
computer
processor to generate instructions to (1) fill a chamber with the medium and
remove the medium
comprising the compound, (2) rinse the chamber to avoid carry-over between
reactions and (3)
repeat (1)-(2) in the same chamber until receiving an input for stop.
[0199] In some embodiments, the first reagent is different from the second
reagent. In some
embodiments, the first reagent is the same as the second reagent.
[0200] In some embodiments, the first chamber and the second chamber comprise
a substantially
same temperature. In another embodiments, the first chamber and the second
chamber comprise
different temperatures.
[0201] In some embodiments, the first chamber and the second chamber comprise
a substantially
same reaction time. In some embodiments, the first chamber and the second
chamber comprise
different reaction times.
[0202] In additional embodiments, the at least one robotic arm is programed to
load the reagents
to the same chamber more than once, and the reagents are all different from
each other, before
loading the reagent to the next chamber. In some embodiments, the at least one
robotic arm is
programed to load the medium with at least one reagent to a chamber before the
next chamber is
loaded in a sequential manner. In some embodiments, the at least one robotic
arm is programed to
load the medium with two reagents to a chamber before the next chamber is
loaded in a sequential
manner. In some embodiments, the at least one robotic arm is programed to load
the medium with
five or more reagents to a chamber before the next chamber is loaded in a
sequential manner.
[0203] Described herein are various methods of use of chambers 1101 as
depicted in FIG. 11. In
some embodiments, the method may comprise the use of a plurality of chambers
1101, wherein at
least one chamber of the plurality of chambers 1101 may remain empty. In some
embodiments, the
method of use of a chamber 1101 comprises a production process. In some
embodiments, the
method of use comprises transporting the cartridge 1200 (as depicted in, e.g.,
FIG. 12A) to a
location of a production system. In some embodiments, the method comprises
transferring the
cartridge from a first location to a second location.
[0204] Described herein are various methods utilizing the systems and devices
described
elsewhere herein in accordance with some embodiments. In some embodiments, the
method may
comprise producing and/or synthesizing a nucleic acid. In some embodiments,
methods may
comprise producing and/or synthesizing RNA. In some embodiments, methods may
comprise
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producing and processing biomolecules such as DNA, RNA, and protein. In some
embodiments,
the method may comprise in vitro transcription of RNA. In some embodiments,
the method may
comprise mixing IVT reagents in a premix. In some embodiments, methods
utilizing the chambers
and/or cartridges may perform the IVT reaction. In some aspects, chambers or
cartridges in
5 accordance with some embodiments, may be used for downstream processing
of RNA. In an
embodiment the chambers and cartridges may be used for post-transcriptional
capping of RNA. In
another embodiment the chambers and cartridges may be used for co-
transcriptional capping of
RNA.
[0205] Alternatively, the chambers or cartridges as disclosed herein may be
used for DNA
10 synthesis. In some embodiments, the chambers or cartridges may be used
for downstream
processing of DNA after synthesis. In some embodiments, the method may
comprise a DNA
synthesis method comprising thermocycling. In some embodiments, chambers or
cartridges in
conjunction with the DNA-preparation method for plasmid amplification may be
used, for
example.
15 [0206] Described herein are various embodiments of a method for
separating and/or purifying a
compound, using devices and the systems disclosed herein. In some embodiments,
the method
comprises: (a) providing one or more sample containers comprising a compound
and magnetic
particles in a liquid medium, wherein each sample container is positioned in a
sample holder; (b)
allowing the compound to mix with the magnetic particles by mechanical motion
of the sample
20 holder; (c) capturing or introducing a movement of the magnetic
particles towards a magnet unit
present in the vicinity of the sample holder, thereby causing a separation of
the liquid and the
magnetic particles; and (d) removing the liquid. In some embodiments, the
method further
comprises adding a liquid to the magnetic beads. In some embodiments, the
method further
comprises repeating (b) to (d). In a further embodiment, (b) to (d) are
repeated at least 2 to 10
25 times, 2 to 8 times, 2 to 7 times, 2 to 6 times, 2 to 5 times, 2 to 4
times, or 2 to 3 times, such as at
least 2 to 5 times. Alternatively, (b) to (d) are repeated at least 3 to 10
times, 4 to 10 times, 5 to 10
times, 6 to 10 times, 7 to 10 times, 8 to 10 times, or 9 to 10 times.
[0207] Described herein are various embodiments of a method comprising:
providing or obtaining
the chamber as described in any one of the preceding claims; and producing a
nucleic acid. The
30 method of any one of the preceding claims, wherein the nucleic acid is a
ribonucleic acid (RNA).
Example Reactor Systems
[0208] In some embodiments, as shown in FIG. 1, the chambers 113 are arranged
in a circular
manner, divided from each other by upstanding walls. In some embodiments, as
illustrated in
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FIGS. 2 and 3, the chambers are respectively arranged in a rectangular manner
or in a honeycomb
structure.
[0209] In some embodiments, the chambers comprise an extended body with a
particular cross-
sectional geometry, such as those depicted in FIGS. 11 and 12A-12J.
[0210] In further embodiments, the chambers may be comprised in cartridges,
such as those
depicted in FIGS. 12A-12J.
[0211] In some embodiments, as shown in FIGS. 1 and 2, the chambers are not in
fluid connection
with each other. The chambers are thus designed to allow retrieving and
holding multiple reagents
by means of a robotic arm located in the vicinity of said cartridge and
chambers. In FIG. 1, the
robotic arm 112 is located in a middle opening of the cartridge. In some
embodiments, as shown
in FIG. 2, the robotic arm 222 is located above the chambers 220 disposed in
the cartridge 221, or
can be installed next to the cartridge (not shown). It will be clear that
other arrangements not
depicted in the Figures equally fall within the scope of the current
invention.
[0212] Each chamber is designed to receive at least one reagent or multiple
reagents via a first
opening. In some embodiments, a second opening may be present, different from
the first opening,
for the evacuation of the content of said chambers. In some embodiments, the
chamber content
may be evacuated by opening or retracting fully or partially the bottom of the
chamber, causing
the chamber content to flow downwards, for instance to a reservoir or a
channel. In another
embodiment, each chamber may be equipped with channels or tubing allowing the
evacuation of
the chamber content. The chambers are thus designed to behave as small reactor
vessels, for the
production of biochemical molecules. The volume of each chamber is preferably
between 1 and
50 ml, although the dimensions can be adapted depending on the intended use.
[0213] The chambers may be provided by a cover or lid, to prevent unwanted
components from
entering said chamber (e.g., RNases) and from shielding the content of said
chamber from the outer
environment. Said lid or cover may cover the first and possibly also the
second opening. In an
embodiment, the cover or lid is a pierceable membrane or provided with a valve
or inlet for
allowing the entrance of reagents and/or the robotic arm.
[0214] In an alternate embodiment, two or more chambers of the cartridge may
be in fluid
connection with each other. For example, the chambers 330 positioned in the
outer ring of the
cartridge 331 of FIG. 3 may be provided with an opening to allow fluid to flow
into the
corresponding chambers in the inner ring. An appropriate means to transfer the
fluid from one
chamber to the corresponding chamber, could be to tilt the chambers in the
outer ring, causing the
liquid to flow in the corresponding chambers in the inner ring. This
evacuation must be conducted
before the robotic arm resupplies the chambers in the outer ring with
reagents. The honeycomb
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structure has as an advantage that it is more compact than, for instance, the
embodiment shown in
FIG. 1.
[0215] The robotic arm may be equipped with (micro)pipettes or tips for
delivering the reagents
to the chambers. In an embodiment, the pipettes or tips may be removable
and/or single-use.
Required reagents can be delivered by the robotic arm to the chambers. To that
purpose, in an
embodiment the head of the robotic arm is movable from a reagent reservoir to
the opening of the
chambers. Process conditions in these chambers, such as temperature, pH,
reaction speed, etc. can
be closely monitored by a process controller (not shown).
[0216] This set-up allows for the design of the overall system which requires
fewer upscaling steps
and guarantees a high quality biomolecule product due to the strict process
control. Furthermore,
the small size of the system facilitates operation in an isolator, a biosafety
cabinet, or other
compartment, such the one as shown in FIGS. 9A-9B.
[0217] In another embodiment, as shown in FIG. 1, the chambers 113 comprise a
single opening
through which the reagents are administered, and the products are evacuated.
These operations are
conducted by the robotic arm 112 in the middle of the carousel 100. The
robotic arm 112 rotates
from one chamber 113 to the next, each time first evacuating the content of
the chamber,
rinsing/washing the chamber and subsequently administering the reagents again,
thereby, assuring
a semi-continuous product flow.
[0218] FIG. 2 illustrates another embodiment of a cartridge 221 wherein the
chambers 220 are
positioned in a rectangular manner, divided from each other by upstanding
walls. The robotic arm
222 is positioned directly above the chambers but could also be positioned in
the vicinity of the
cartridge. As for the embodiment of FIG. 1, the robotic arm 112 serves to add
reagents to openings
of the chamber 113. Evacuation of the content of the chambers 113 may be
performed by the same
robotic arm 112, or a second robotic arm (not shown). In another embodiment,
evacuation may
occur via a second opening, configured to evacuate the content of one or more
chamber. Said
second opening could be thus positioned that it allows evacuation from
underneath said chamber.
[0219] FIG. 3 further illustrates yet another embodiment of a cartridge 331
wherein a cartridge
331 comprises chambers 330 in a honeycomb structure. Again, a robotic arm 332
is positioned in
the vicinity of the cartridge 331 such as in an opening in the middle of
cartridge 331, allowing
manipulation of said chambers 330 (adding of reagents and/or evacuating
content).
[0220] In an embodiment, the chambers 330 as shown in FIG. 3 are not in fluid
connection with
each other. However, in an alternate embodiment, two or more chamber of the
cartridge may be in
fluid connection with each other. For example, the chambers 330 positioned in
the outer ring of
the cartridge 331 of FIG. 3 may be provided with an opening to allow fluid to
flow into the
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corresponding chambers in the inner ring. An appropriate means to transfer the
fluid from one
chamber to the corresponding chamber, could be to tilt the chambers in the
outer ring, causing the
liquid to flow in the corresponding chambers in the inner ring. This
evacuation must be conducted
before the robotic arm resupplies the chambers in the outer ring with
reagents. The honeycomb
structure has as an advantage that it is more compact than, for instance, the
embodiment shown in
FIG. 1.
[0221] FIGS. 1 to 4 show only one robotic arm but it will be appreciated by
those skilled in the
art that multiple robotic arms can be present, for instance when different
reagents are to be provided
by different robots or when evacuation of the chamber content is performed by
a separate robotic
arm.
[0222] In one embodiment, the cartridge may be static. In another embodiment,
as depicted in
FIG. 4, the cartridge 441 may be allowed to perform a movement, such as a
rotation (see arrows
in FIG. 4, indicating the rotation of the cartridge). This embodiment allows
for a more static robotic
arm since the rotational movement of the cartridge 441 brings those chambers
440 that require the
addition of a reagent or evacuation in the vicinity of the robotic arm, rather
than the robotic arm
moving to each of the chambers. The system may thus be programmed such that
the cartridge is
rotated in a predefined manner, such as with a particular rotational speed
(e.g., determined based
at least in part on a reaction/incubation time or number of chambers). As for
the embodiments
shown in FIGS. 1 to 3, the robotic arm may provide reagents to the opening of
the chambers and
may be configured to evacuate the content of the chambers. Alternatively,
evacuation may occur
by means of the second opening of the chamber. This second opening may be
present at the bottom
of each chamber. The second opening could be positioned on the top of the
covering lid, with the
system to suck the liquid, like a dip tube for example.
[0223] A particular embodiment is shown in FIG. 5, wherein the plurality of
cartridges 550 and
robotic arms 551 & 552 are provided. The embodiment of FIG. 5 allows several
cartridges 550 to
be worked on in parallel, which may enhance the output and the efficiency of
the production of
biochemical molecules. Alternatively, the configuration shown in FIG. 5, or
variants thereof, may
be used to perform a sub-reaction in a first cartridge, after which the
content of the first cartridge
is transferred to a second cartridge for further reaction. Meanwhile, the
first cartridge and its
chambers may be provided with reagents to start a new sub-reaction. Again, the
design in FIG. 5
may enhance the final output of the system.
[0224] In some embodiments, transfer of content from a first to a subsequent
cartridge and/or
corresponding subsequent chamber may be performed by the robotic arm. In
another instance, the
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embodiment as shown in FIG. 5 and variants thereof may be equipped with piping
and pumps to
transfer the content from the cartridges and/or chambers.
[0225] In some embodiments, as mentioned, the design as shown in FIG. 5
comprises several
cartridges which can be easily combined. This may, for instance, serve to
increase the production
volume in a particular amount of time.
[0226] FIG. 6 illustrates another embodiment of a cartridge according to the
current disclosure,
showing a cross-sectional view of a cartridge, having multiple chambers 661,
662, 663, and 664 in
a vertical arrangement, each provided with an inlet for reagents. In the
embodiment shown in FIG.
6, each chamber 661, 662, 663, and 664is at its bottom surface equipped with a
valve, allowing the
transfer of fluid content from a first upper chamber to a lower positioned
second chamber by means
of gravitation. When transfer of liquid is desired, one or more valves may
receive the instruction
to open to allow fluid flow. Similarly, the valves may also be instructed to
close, in order to allow
a (sub)-reaction in the chamber. In the embodiment shown in FIG. 6, the bottom
chamber 664 of
the cartridge is provided with an outlet 665 for the evacuation of the content
of the lower chamber.
In some embodiments, the outlet 665 may be provided with one or more pipes to
allow transfer of
the fluid to another compartment, such as a manifold or to a downstream
processing unit (not
shown on FIG. 6). In some embodiments, the cartridge may be thus designed to
allow sub-
reactions taking place in one chamber, and once the sub-reaction is performed,
the content of a
chamber will be transferred to a lower residing chamber, allowing the further
addition of reagents
via the chamber inlets. Chambers that have been evacuated are then ready to
obtain a subsequent
sub-reaction. As such, sequential production of biochemical molecules can take
place.
[0227] In some embodiments, the top chamber 661 of the cartridge can be
provided by an inlet
666 for reagents on a top surface of the cartridge, or any other suitable
position. It will be
appreciated by a skilled person that not all chambers require the presence of
inlets. In some
embodiments, only the top chamber 661 can be provided by an inlet 666, whereas
the bottom
chamber 664 is provided with an outlet 665. In an embodiment, a feedback-loop
from the outlet of
the bottom chamber 664 can be provided, to one of the above positioned
chambers, such as the top
chamber 661.
[0228] While FIG. 6 shows one embodiment allowing the transfer of liquid from
one chamber to
another, it will be appreciated that other means of fluid transfer are also
possible. Such means,
although not shown in the Figures, may comprise: a tubing between two
successive chambers and
a pumping system to move the liquid; a pressure system in each chamber and
valves at the inlet
and at the outlet of each chamber; a mechanical system which lifts each
chamber and rotate each
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chamber to flow the liquid in the next chamber; a variable angular inclined
plane; or any
combination thereof
[0229] FIG. 7 shows another embodiment of the cartridge 771, comprising the
plurality of
chambers (shown in cross section). In some embodiments, the chambers are
configured to move
5 on a conveyor belt 772 powered by one or more powered pulleys 773 & 774.
In some embodiments,
one pulley may be powered and one may be idle. In such cases, the former may
be referred to as
the drive pulley while the latter, unpowered pulley, may be referred to as the
idler pulley.
[0230] A robotic arm 775 is provided to allow transfer of reagents to and from
the chambers. A
reaction may be started in a first position when the robotic arm 775 provides
the necessary reagents
10 to the chamber. The chambers are then transferred by traversing them in
one or more directions
until they occupy a second position. In the second position, the content of
the chamber can then
be removed gravitationally from the chambers. To enable reloading, the chamber
is then transferred
along the conveyor 772 from the second position back to the first position.
The time between
evacuation and reloading can be used for, e.g., disinfection and/or cleaning
of the chambers.
15 [0231] FIG. 8 shows a conceptual representation of the downstream
processing steps that occur
after one or more reactions have taken place in the cartridge 881. The result
of the multiple
reactions taking place in the chambers 880 is collected in a manifold. This
can be done by any
suitable method . The manifold 882 serves as an intermediate vessel to collect
several volumes
coming from the cartridge 881 and the chambers 880. Once a predefined or
sufficient volume is
20 collected in the manifold 882, the volume will then be processed in a
downstream processing unit.
Such downstream processing unit may comprise one or more purification devices
883 suited for
purification of the product produced in the cartridges 881 and collected in
the manifold 882. The
downstream processing may be a batch process, such as batch purification. In
another embodiment,
it may also be a continuous mode process. Sufficient transfer means such as
tubing, valves and
25 pumps may be present to allow transfer of liquid to the manifold and
from the manifold to the
downstream processing unit.
[0232] The cartridges, manifold and downstream processing unit as described
above may be
embedded in a biomolecule production system. An example of such system is
shown in FIGS. 9A-
9B. The example system is a concatenation of different modular, optionally
movable, units in
30 which different steps of the biomolecule manufacturing process are
conducted. A modular unit
comprises the appropriate devices and equipment for performing part or the
complete biomolecule
manufacturing process. In one embodiment, the equipment for upstream
processing such as
cartridge, robotic arm, and manifold may be provided in one unit whereas the
required downstream
processing devices are present in a separate unit. These units can be
connected to each other.
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[0233] In an embodiment, the different units may be provided with a connector,
allowing the
connection of the units. The connector can be configured to allow physical
combination of different
units by a modular connector system allowing the transfer of data. To that
purpose, said connector
may comprise combinations of power and signal contacts, Ethernet, optical
fiber, coaxial contacts,
hydraulic, pneumatic and thermocouples in a compact frame or housing. This
modular connector
system can be configured according to the specific requirements of the
connection. The connectors
can be waterproof In an embodiment, one unit can be provided with a male
connector, connectable
to a female connector of a second unit. To ensure correct connection between
the male and female
connector, the female connector may contain centering pins. In another
embodiment, the connector
comprises an electronic eye to ensure correct connection. In another
embodiment, the connector
comprises magnetic elements to ensure correct connection.
[0234] In an embodiment, the connector is configured to couple the bioreactor
cabinet to a
bioreactor chamber of the production system by means of a connecting portion
and receiving
portion. The connecting portion may be located at the bioreactor cabinet
whereas the receiving
.. portion may be present in the bioreactor chamber of the system or vice
versa.
[0235] In a further or in another embodiment, a connecting portion on a first
unit and a receiving
portion on a second unit will allow docking of both units to ensure that both
entities are firmly
connected to each other, prohibiting the release of a first unit from a second
or further unit during
the production of biomolecules. This connecting and receiving portion can be
any suitable
connecting system, such as of mechanical or magnetic system. A break-away
function can be
incorporated to be able to release the units.
[0236] In an embodiment, a unit may be comprised of both a connector allowing
the transmission
of power, signals, and/or data when paired with another unit and a magnetic
connection that allows
docking of a first unit to a second or subsequent unit. In an embodiment, said
magnetic connection
may be a permanent magnet. In another embodiment, said magnet may be an
electromagnet,
wherein a magnetic field is produced by an electric current. The electromagnet
may allow the
magnetic field to be quickly changed by controlling the amount of electric
current. In some
embodiments, the use of a magnet, such as an electro-magnet, enhances the
safety of the system,
as it may prevent unauthorized docking or removal of units. A second or
subsequent unit may be
.. comprised of a corresponding magnetic part to allow interaction with the
magnet of a first unit.
[0237] The system may be provided with separate units for the delivery of
reagents and the
evacuation of waste, as well as for the collection of the biomolecule harvest.
Each unit is thus
designed to allow the provision of filtered, sterile air to be circulated
within the units. Air filtering
means may include for instance a HVAC system with HEPA filters.
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[0238] The use of different modular units allows for the system to be
assembled or constructed on
site rapidly, and potentially disassembled with similar rapidity. As it is
possible to easily add
additional modular units, the number of cartridges, manifolds or purification
units may be adjusted
to suit a particular need or process setting depending on the application.
.. [0239] In the embodiment shown in FIGS. 9A and 9B, a central unit 990is
equipped with a docking
station 991 for receiving a cartridge 992 and optionally a robotic arm 993.
The robotic arm 993
and the operating system may alternatively be present in the central unit 900.
The central unit 900
is further provided with the appropriate equipment or docking places 994 for
downstream
processing of the harvest from the cartridge (e.g., chambers, cartridges,
and/or robotic arms). As
shown in FIG. 9A, the central unit may be coupled to accessory units 995
(e.g., fridges), for
instance for the provision of the necessary reagents, the collection of the
end product, storage of
reagents or intermediates, and/or the evacuation of waste.
[0240] One or more units may be mobile and, in such cases, provided with
transportation means
that allow the mobility or transportation of said bioreactor cabinet For
example, in the system
depicted in FIG. 9A, each accessory unit 995 is provided with wheels 996.
Handles 997may also
be present, for easy handling of the accessory unit(s). The handle(s) make(s)
it possible for an
operator to move the bioreactor cabinet by pushing or pulling on the
handle(s). The handle(s) can
have various sizes and can be placed at different positions on the outside of
a unit or portion thereof.
[0241] The housing of the units may be made of any suitable material, such as
metal alloy, metal,
or plastic. In one embodiment, a unit is made from a material comprising
aluminum or stainless
steel. In another embodiment, the bioreactor cabinet is made of a material
comprising stainless
steel.
[0242] The units can be designed and operated such that they only require
limited handling of the
operator. This is to avoid contamination and disturbance of the process
conditions. If irregularities
are observed, the operator can manipulate the process via one or more control
devices present
inside or outside the unit(s). These control devices control (e.g., parts of)
the process taking place
in the unit. Each unit may be coupled to one or more control devices that are
configured to perform
multivariate analysis, automatically control operation of the processes, and
optionally,
communicate with components remotely (using, for example, network protocols)
in order to
control operation in the unit(s).
[0243] In some embodiments, a chamber 1101 used in the methods and systems
described herein
comprises one or more features as shown in FIG. 11. In some embodiments, the
chamber 1101
comprises six flat, polygonal (e.g., rectangular) surfaces 1104. In some
embodiments, the chamber
comprises a cross section, the cross section (e.g. transversal section)
comprising a hexagonal shape.
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In some embodiments, the chamber 1101 comprises a lid 1102, the lid 1102
comprising an opening
1103. In some embodiments, the lid 1102 comprises a push-on lid. In some
embodiments, the
opening 1103 is configured to allow for access to the chamber 1101. In some
embodiments, the
opening 1103 is configured to allow for access, to provide contents to the
chamber 1101. In some
.. embodiments, as shown in FIG. 11, the lid comprises a hexagonal shape. In
some embodiments,
the chamber 1101 does not comprise a lid.
[0244] In some embodiments, as shown in FIG. 11, the bottom 1105 of the
chamber is pointed. In
some embodiments, the bottom 1105 may be flat. In some embodiments, the bottom
1105 may be
rounded (e.g., concavely or convexly).
[0245] In some embodiments, chambers 1101 are comprised in one or more
cartridges 1200, such
as those depicted in FIGS. 12A-12J. In some embodiments, the cartridges may
comprise a plurality
of chambers 1101 as shown in FIGS. 12A-12J. In a non-limiting example shown in
FIG. 12A,
the cartridge 1200 comprises 4 rows of chambers 1101. In another non-limiting
example depicted
in FIG. 12C, the cartridge 1200 comprises 3 rows of chambers 1102. In some
embodiments (e.g.,
as depicted in FIGS. 12A-12J), outer rows of chambers 1101 of the cartridge
1200 comprise the
highest number of chambers 1101. In some embodiments, the middle rows of
chambers 1101
contain fewer chambers 1101 than the outer rows of chambers 1101. The
cartridge 1200 shown in
the non-limiting example of FIG. 12B comprises chambers comprising a larger
volume than the
chambers shown in the non-limiting example of FIG. 12A. However, the volume of
a chamber
1101 may comprise any suitable value, such as those disclosed elsewhere
herein. In some
embodiments, the chamber comprises a volume of about 50 mL. In some
embodiments, the
chamber comprises a volume of about 20 mL. In some embodiments, the chamber
comprises a
volume of about 10 mL. In some embodiments, the chamber comprises a volume of
about 5 mL.
In some embodiments, the chamber comprises a volume of about 4 mL. In some
embodiments, the
chamber comprises a volume of about 3 mL. In some embodiments, the chamber
comprises a
volume of about 2 mL. In some embodiments, the chamber comprises a volume of
about 1 mL.
[0246] In some embodiments, the cartridge 1200 comprises an aperture 1201 as
shown in the non-
limiting examples of FIG. 12A and 12B. In some embodiments, the cartridges
1200 comprise at
least one aperture. In some embodiments, the cartridge 1200 comprises at least
two apertures 1201.
In some embodiments, the cartridge is configured to engage with a gripping
mechanism of a robotic
arm or handling device. In some embodiments, (such as those depicted in FIGS.
12C, 12D, 12E,
and 121), the cartridge may comprise one or more markers 1202 to aid in
positioning of the
cartridge, such as within a modular unit (e.g., carousel, conveyor, heater,
shaker, or combination
thereof) as described above.
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[0247] In some embodiments, such as those depicted in, e.g., FIGS. 12F and
12J, at least a subset
of the chambers share an edge or vertex with another chamber. In some
embodiments, such as
those depicted in, e.g., FIGS. 12A-12E, none of the chambers shares an edge or
vertex with another
chamber.
[0248] In some embodiments (such as those depicted in FIGS. 12A-12B), the
chambers comprise
rounded bottoms. In some embodiments (such as those depicted in FIGS. 12C-
12J), the chambers
comprise pointed bottoms.
[0249] The embodiments and devices as described above may be used in the
manufacturing of
biologicals. In one embodiment, devices as described herein above are used in
the production of
.. RNA by means of an in vitro transcription (IVT) reaction. Alternatively, or
in a further
embodiment, the embodiments may also be used for the cell-free production of
protein starting
from RNA (or a DNA strain that is to be transcribed). In some embodiments, the
production of a
biological comprises a cell-based production of protein, using, e.g., an
inoculum of transiently
transfected cells.
[0250] An IVT reaction as described herein may generally comprise one or more
operations of:
mixing a DNA template with nucleotides, DNA polymerase and other reagents; and
incubating the
mixture at a defined temperature for a defined duration during which: the DNA
polymerase "reads"
the DNA template and catalyzes the synthesis of the corresponding RNA
molecule; the RNA
molecule is provided with a capping structure at its 5' end by either: co-
translational capping
(whereby specific reagents are introduced into the reaction mix at the start
of the reaction); or post-
transcriptional capping (= transformation, by enzymatic action, of the
starting nucleotide in the
already formed RNA molecule to a capping structure comprising N7-
methylguanosine linked to
the starting nucleotide by a triphosphate). In some embodiments, an IVT
reaction comprises a
subset of these operations. In some embodiments, an IVT reaction comprises
additional operations
(e.g., downstream purification and/or isolation).
[0251] Finally, and depending on the design of the process, the transcription
reaction might be
followed by enzymatic digestion of the DNA template, facilitating downstream
removal of DNA.
[0252] The current disclosure and its embodiments allow for the sequential
production of small
volumes in the chambers of a cartridge, which may subsequently be combined and
processed
downstream. In an embodiment, a chamber of a cartridge can be provided with
reagents for
allowing the production of biomolecules. A second chamber can, within a time-
interval after the
first chamber has been provided with reagents, also be provided with reagents.
Eventually, a chain
of reactions will be happening in each of the chambers, each reaction having a
different starting
and endpoint. Consequently, a virtual 'endless' process of production can be
achieved, wherein
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between specific time-intervals the output of a chamber can be harvested and
combined with the
output of other chambers.
[0253] Finally, after production and downstream purification, the bulk drug
substance is
formulated and filled in individual containers (with or without additional
processing steps such as
5 lyophilization) to form the drug product.
Examples
Example 1: Assessing impact of rerunning reaction in the same chamber
[0254] An experiment was performed in a container as described herein
comprising 21 chambers,
6 of which were used for the experiment. To evaluate the reusability of the
chambers, the IVT
10 reactions were performed 12 times with washing (2 x 1 mL water) in
between each reaction. An
overview of the reaction set-up is depicted in FIG. 13. Two chambers (samples
1 and 2 of FIG.
13) contained reagents and enzymes for IVT (DNA, enzymes, NTPs, CleanCap
reagents, and
buffer). Two chambers (samples 3 and 4 of FIG. 13) were negative controls with
reaction mixtures
comprising nuclease-free water (WFN), glycerol, and buffer¨but no DNA,
enzymes, NTPs, and
15 CleanCap reagents. Two chambers were blanks comprising only WFN. In
parallel, the same IVT
reaction was performed in a series of Eppendorf tubes as controls. A positive
control (IVT reaction,
sample 7 in FIG. 13) was performed in a new tube each cycle. Another set of
controls (samples 8
and 9 in FIG. 13) comprised the IVT reaction performed in the same tube each
time with washing
as described above in between each cycle. Finally, two tubes (reactions 10 and
11 in FIG. 13)
20 comprised negative controls corresponding to samples 3 and 4, except
performed in Eppendorf
tubes (including washing and reuse between each IVT cycle).
[0255] An overview of the IVT reaction is shown in FIG. 14. The pre-mix was
prepared and
dispatched in the corresponding cartridges and Eppendorf tubes. The pre-mix
was preheated for 10
min. at 45 C before the addition of T7 RNA Polymerase (T7 Pol) mix to start
IVT. IVT was
25 allowed to proceed for 75 min with constant agitation and the reaction
mixtures open to the
atmosphere. Following 75 min of IVT, the contents of each IVT well/tube was
transferred to a new
Eppendorf tube for DNase treatment and quenching. Each IVT well/tube was then
washed two
times with 1 mL of water. Each wash was retained for UV-Vis spectrophotometry
to evaluate
possible adsorption of compounds from the reaction mixture (e.g., nucleotides,
nucleic acids,
30 proteins) to the reactor or tube wall due to repeated use.
[0256] To determine changes in mRNA production as a function of cycle, the
amount of mRNA
produced by each reaction in each cycle was measured using a Qubit
fluorescence assay. The
results of the measurements are shown in FIG. 15A. The ratio of mRNA produced
in the chamber
or reused Eppendorf tube to the corresponding new Eppendorf tube was also
calculated for each
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reaction at each cycle and is shown in FIG. 15B. As shown by FIGS. 15A-15B,
the amount of
mRNA produced did not appear to vary as a function of the number of IVT
reactions that had been
performed in a given chamber.
[0257] Additionally, the two washes of each cartridge were assayed by UV-Vis
spectrophotometry
(NanoDrop) to determine how much residual nucleic acid and/or protein was
removed by washing.
As shown in FIGS. 16A-16B, two washes were sufficient in each case to remove
residual nucleic
acids (260 nm, FIG. 16A) and proteins (280 nm, FIG. 16B).
[0258] Integrity of the mRNA produced was measured as a function of reaction
cycle by subjecting
the synthesized RNA to fragment analysis by capillary gel electrophoresis
after reactions 1, 6, and
12. The data are shown in Table 1. The area and relative concentration of the
main peak was found
to be about constant over each measurement, suggesting that the target
molecule was made in each
case and that reuse of the cartridges did not impact integrity of the
synthesized RNA.
Table 1. Fragment analysis of synthesized RNA
IV Npeaks Npeak HighestPea HighestPea 2n 2nd 3r 3rd 4t 4th % Aver
%C
T tot. >1% kPerc kSize d size d size h Size Purity age
V
Prototype
6 1331 4 2521 2 860 91.5
R1 4 92 1 987
91 6
0.2
Prototype
7 1318 4 2494 2 852 91.7
R2 4 92 1 979
Eppendoif
1 7 1331 4 2548 2 856
92.1
R1 4 92 1 987
924
0'5
Eppendoif '
%
6 1325 4 2521 2 852 92.7
R2 4 93 1 979
Eppendoif
N/
6 1325 4 2507 2 852 92.4 924 A new 4 92 1 987
Prototype
7 93 1325 4 2507 2 856 92.6
R1 4 1 983
92.8
0'3
Prototype
%
5 1331 4 2521 2 856 93.0
R2 4 93 1 987
Eppendoif
6 7 1331 3 2507 2 856 92.4
R1 4 92 1 983
928
0.5
Eppendoif '
%
6 1338 3 2535 2 860 93.1
R2 4 93 1 992
Eppendoif
N/
6 1325 4 2507 2 852 92.5 92'5 A new 4 93 1 983
Prototype
7 92 1331 4 2521 2 856 91.5
R1 4 1 983
91.9
0'5
Prototype
%
6 1336 3 2529 2 864 92.2
R2 4 92 1 987
Eppendoif
12 5 93 1322 3 2515 2 859 93.2
R1 4 1 983
93 3
0.1
Eppendoif
6 1322 3 2529 2 855 93.3
R2 4 93 1 983
Eppendoif
N/
6 1329 2 2500 2 859 93.4 93'4 A
new 4 93 1 987
[0259] Finally, the relative proportion of double stranded RNA (dsRNA) in each
sample was
calculated after the 15t, 6th, and 12th IVT reactions by and enzyme-linked
immunosorbent assay
(ELISA). The results are shown in FIG. 17. The amount of dsRNA in each sample
was seen to
stay below the upper tolerance limit of 300-500 ng of dsRNA per mg of total
RNA.
Example 2. Computer Simulation of Reactor System
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[0260] To model kinetic properties of a reactor system as described herein and
demonstrate a
continuous cycle of compound production, a computer simulation of the reactor
system was
performed in Microsoft Excel. The parameters of the simulation are as follows:
40 chambers of 20
mL working volume each, giving a total working volume of 800 mL; time interval
between starting
reactions (e.g., by adding a reagent) in subsequent chambers of 4.5 min. (time
interval X = 4.5
min); time interval between start of reaction (e.g., addition of reagent) and
harvesting of chamber
contents (e.g., synthesized compound) of 180 min (time interval Y = 180 min);
time interval
between emptying contents of a chamber and adding new working volume to start
a new reaction
in the same chamber of 3.25 min. The number of chambers corresponds to the
time interval Y
divided by the time interval X (180/4.5 = 40). In practice, the addition and
removal of the working
volume from each chamber may be performed by devices and methods described
herein above,
such as one or more robotic arms configured to add and/or remove working
volume from the
chambers or a subset thereof
[0261] A graph of two reaction cycles in two subsequent chambers is shown in
FIG. 18A. The
first cycle in the first chamber began at time = 0 and lasted for 180 min
(time interval Y). The first
cycle in the second chamber began at time = 4.5 (time interval X) and also
lasted for 180 min. The
first cycle in the first chamber ended at 180 min, at which point the contents
of the first chamber
were harvested and new contents were introduced to begin the second cycle in
the first chamber.
The time delay between the end of the first cycle and the start of the second
cycle in the first
chamber was 3.25 min, such that the second cycle in the first chamber began at
time = 183.25 min.
Analogously, the first cycle in the second chamber ended at time = 184.5 min,
and the second cycle
in the second chamber began at time = 187.25 min. As such, at all times shown
in FIG. 18A the
gap between the two curves is 4.5 min, corresponding to the time interval X.
Although only three
cycles (time period of at least 3Y) are depicted, this process may continue
for as many cycles
desired/possible (e.g., considering the need to halt production to
periodically maintain the system).
[0262] Turning from individual chambers to the bulk system level, FIGS. 18B
and 18C depict the
total working volume in the system at a certain time and the cumulative volume
harvested up to a
certain time, respectively. As may be seen in FIG. 18B, once a time interval Y
had passed, all
chambers in the system were in use, allowing the total working volume to
remain about constant
at about 800 mL (with some fluctuations on the order of the chamber volume
(about 20 mL) due
to (i) the time interval between starting reactions in subsequent chambers
(time interval X) and (ii)
the delay between harvesting the reaction mixture following one cycle and
adding the new reaction
mixture for the next cycle). Further, as depicted in FIG. 18C, once the total
working volume (800
mL) was in full use (after one cycle at time = 180), there was essentially
continuous harvesting of
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material from the chambers. The reactor system, following a transient
"priming" period
corresponding to time interval Y, thus entered into a steady-state
"production," or "running,"
period during which the compound was synthesized essentially endlessly at an
about linear rate,
with no break in operation of the system.
.. [0263] The foregoing has been presented for purposes of illustration and
description. It is not
intended to be exhaustive or to limit the embodiments to the precise form
disclosed. Obvious
modifications and variations are possible in light of the above teachings. All
such modifications
and variations are within the scope of the appended claims when interpreted in
accordance with
the breadth to which they are fairly, legally and equitably entitled.
[0264] While preferred embodiments of the present invention have been shown
and described
herein, it will be obvious to those skilled in the art that such embodiments
are provided by way of
example only. It is not intended that the invention be limited by the specific
examples provided
within the specification. While the invention has been described with
reference to the
aforementioned specification, the descriptions and illustrations of the
embodiments herein are not
meant to be construed in a limiting sense. Numerous variations, changes, and
substitutions will
now occur to those skilled in the art without departing from the invention.
Furthermore, it shall be
understood that all aspects of the invention are not limited to the specific
depictions, configurations
or relative proportions set forth herein which depend upon a variety of
conditions and variables. It
should be understood that various alternatives to the embodiments of the
invention described herein
may be employed in practicing the invention. It is therefore contemplated that
the invention shall
also cover any such alternatives, modifications, variations or equivalents. It
is intended that the
following claims define the scope of the invention and that methods and
structures within the scope
of these claims and their equivalents be covered thereby.
