WO2022112416A1 - Nouveau lit de chromatographie - Google Patents

Nouveau lit de chromatographie Download PDF

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Publication number
WO2022112416A1
WO2022112416A1 PCT/EP2021/082999 EP2021082999W WO2022112416A1 WO 2022112416 A1 WO2022112416 A1 WO 2022112416A1 EP 2021082999 W EP2021082999 W EP 2021082999W WO 2022112416 A1 WO2022112416 A1 WO 2022112416A1
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Prior art keywords
bed
chromatographic system
chromatographic
channels
porous
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PCT/EP2021/082999
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English (en)
Inventor
Alois Jungbauer
Peter SATZER
Gregory Silva DUTRA
Original Assignee
Universität Für Bodenkultur Wien
Acib Gmbh
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Publication of WO2022112416A1 publication Critical patent/WO2022112416A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/261—Synthetic macromolecular compounds obtained by reactions only involving carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28042—Shaped bodies; Monolithic structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28042—Shaped bodies; Monolithic structures
    • B01J20/28045—Honeycomb or cellular structures; Solid foams or sponges
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28052—Several layers of identical or different sorbents stacked in a housing, e.g. in a column
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
    • B01J20/28095—Shape or type of pores, voids, channels, ducts
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
    • B01J20/28095—Shape or type of pores, voids, channels, ducts
    • B01J20/28097—Shape or type of pores, voids, channels, ducts being coated, filled or plugged with specific compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/305—Addition of material, later completely removed, e.g. as result of heat treatment, leaching or washing, e.g. for forming pores
    • B01J20/3064—Addition of pore forming agents, e.g. pore inducing or porogenic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/52—Sorbents specially adapted for preparative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/80—Aspects related to sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J2220/82—Shaped bodies, e.g. monoliths, plugs, tubes, continuous beds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N2030/524—Physical parameters structural properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N2030/524—Physical parameters structural properties
    • G01N2030/528—Monolithic sorbent material

Definitions

  • the present invention relates to the field of chromatography bed design and manufacturing and use thereof.
  • Packed bed chromatography is currently the most used technology for biomolecule separation as well as for separation tasks in other industry.
  • the widespread application of this technology led to a huge endeavor for optimization and current chromatography packed bed medias are very suitable for biomolecules, but still fall short for bio-nanoparticles as such as viruses, virus like particles or exosomes, which is especially a problem for novel gene therapy products or vaccines for emerging viral diseases.
  • the main limitation of packed bed chromatography is the unavoidable packing method itself, as the beads have to be packed into a column. The beads do not arrange in a perfectly regular structure during the packing of the column. This packing with chromatography resin beads results in a typical unregular structure of the chromatographic bed.
  • the structure of the chromatography bed is different at the wall and the adjacent layer of beads which often results in so called wall effects.
  • the irregular bed structure and the different structure of the chromatography bed close to the wall lead to a loss of separation efficiency.
  • the lack of ordered structure in the chromatography bed and the wall effect is even more pronounced in smaller columns intended for analytical separation purpose with very narrow pore size.
  • the second technology relates to the use of monolithic structures, where a polymerization mixture including a porogen is introduced into the chromatographic housing, and the structure itself is generated by phase separation and polymerization of one of the phases.
  • This structure is completely open to the convective flow and therefore also eliminates the issue of low diffusivity of larger entities.
  • phase separation and polymerization is an inherently unstructured and irregular process.
  • the distribution of the resulting pore sizes throughout the material is in a broad range. While macroscopically the structure seems to be homogenous, on the microscopic scale it is purely random.
  • monoliths also have an unstructured bed and the problem of wall effects due to pores ending in the wall as well as potential gaps between the monolithic structure and the chromatographic housing.
  • Stacked membranes are also used for separation purposes.
  • Membranes can be equipped with large pores allowing the convective transport of large proteins, polymers and bio-nanoparticles such as viruses, virus-like particles or exosomes.
  • Stacking of membranes generates a high degree of in-homogeneity and the sealing of the gap between the wall and the membranes is extremely critical.
  • membranes themselves consist of irregularly shaped pores. This leads to much lower resolution when compared to monolithic systems and conventional packed chromatographic beds.
  • Projection micro stereolithography (PpSL with a reported resolution of 0.6- 2 pm) can achieve the necessary resolutions, but no commercial instrument is available to generate the object size needed to be connected to a chromatographic system.
  • EP3297757A1 discloses a chromatography column comprising a porous monolithic moulding as sorbent and a cladding, characterized in that the moulding and the cladding have been produced together from the same thermoplastic polymer by means of 3D printing processes.
  • US10493693B1 discloses additively manufactured monolithic structures for containing and directing flows of fluids.
  • W02020089421A1 disclosed is a method for the production of a porous polymer membranes suitable for liquid filtration or analyte capture.
  • a flowable composition comprising photo-activatable monomer molecules, photo activation initiator molecules and photo-activation quencher molecules. The composition is treated with laser light to locally polymerize the composition.
  • W02018005647A1 discloses a methods and device for detecting rare cells in whole blood samples comprising chemically functionalized hydrogel matrix.
  • W02004002603A1 discloses permeable polymeric monolithic materials which are polymerized by applying a pressure. The pressure eliminates wall effect and changes the structure in the column.
  • US2013012612A1 discloses a process for producing three-dimensional, self-supporting and/or substrate-supported formed structures on surfaces by means of site-selective solidification of a liquid to pasty material by means of two- or multiphoton polymerization.
  • the formed surface structures are suitable as matrices for binding live cells.
  • W02017001451A1 relates to photopolymerization methods, specifically to two- photon stereolithography (TPS) which allows the generation of high-resolution 3D arbitrary microstructures. Accordingly, Molecularly Imprinted Polymers (MIP) are prepared by two-photon stereolithography.
  • W0201710863A1 describes a separation medium based on hydrogel.
  • Said separation medium is defined by a triply periodic minimal surface, wherein the channels of the structure are about 5 to 500 pm diameter and the hydrogel has a porosity of about 50 to 90%.
  • US20130075317A1 relates to monolithic porous material made of amorphous silica or activated alumina, wherein the channels have a substantially uniform cross- section which is regular over the entire lengths. The channels are of capillary dimension not exceeding a few millimeters.
  • Conan F. describes packed porous beds for a wide range of chemical engineering unit operations.
  • the printed porous bed consists of discrete particles interconnected forming macrostructures which are in same way akin to monoliths.
  • Moleirinho M.F. et al. describe 3D-printed cellulose chromatographic columns functionalized with two different ligands for purification of viral particles.
  • the solid phase additionally has a porous nature.
  • the channels have a diameter of 300 pm while the pores of the solid phase have size distribution between 10 and 0.5 pm.
  • Salmean C. and Dimartino S. reviewed the state of the art of 3D-printed stationary phases with ordered morphology. Most of the disclosed the structures have been designed hypothetically and have not been put into practice.
  • Two photon polymerization is a technology with a nominal resolution of 0.1- 1 pm and which can achieve the necessary resolutions for chromatographic base material but traditionally has very long printing times of days or maybe even months for objects large enough for chromatography.
  • the technology was advanced by UpNano, being able to generate life size objects (up to 5x5x5 mm) with micro and sub-micro resolution in a matter of hours, making this technology a possible and suitable candidate for the generation of chromatographic bad material.
  • It is the object of the present invention to provide a chromatographic system comprising a chromatographic bed material with strictly regular channels for separation of biomolecules such as proteins and other biopolymers including bio-nanoparticles such as viruses, virus like particles or exosomes.
  • the present invention provides a chromatographic system comprising a porous bed with uniform structure throughout the entire bed material to the sidewall, wherein the porous bed consists of strictly regular straight channels with a diameter of less than 100 pm.
  • the porous bed has strictly regular straight and independent channels, e.g., the channels are not interconnected
  • the bed material is a polymer, a mixture of polymers, or silicate.
  • the polymeric material is selected from the group consisting of poly(meth)acrylate, poly (glycidy I methacrylate), and polymethylmethacrylate.
  • the channel width is in the range of about 0.1 to 100 pm.
  • the walls of the channels are made porous through the use of a porogen.
  • the polymeric material carries functional groups.
  • the surface of the channels possess functional groups.
  • the chromatographic bed is encompassed in a housing with or without flow distributor at one or both ends of the porous bed.
  • the housing is made of steel, of plastics, of the polymer.
  • the present invention further provides an extended chromatographic system comprising two or more porous beds, wherein the beds differ in the functional groups present in the channels.
  • the present invention provides a method for producing a chromatographic system, comprising the steps of: a. providing monomers as base bed material, b. polymerizing the monomers by utilizing two photon polymerization thereby printing a porous bed with uniform structure throughout the entire chromatographic bed to the sidewall, whereby strictly regular straight channels with a diameter of less than 100 pm are produced; c. optionally functionalizing the channels, and d. optionally providing a housing.
  • the porous bed of step and the housing of the chromatographic system are 3D-printed simultaneously as one piece.
  • the chromatographic system is used for separation of nanoparticles, specifically for the separation of bionanoparticles.
  • bionanoparticles are selected from the group consisting of viruses, virus like particles, extra cellular particles, exosomes or other extracellular vesicles, polymer particles with or without being covered by proteins or nucleic acids or inorganic particles with or without being covered by proteins or nucleic acids.
  • a further embodiment relates to the method for separating at least one target nanoparticle as described herein, comprising the steps of: a. providing a chromatographic system as described herein; b. applying a medium comprising the target nanoparticle; and c. eluting the target nanoparticle thereby avoiding band broadening effects.
  • Fig. 1A shows the dimension of printed monolithic structures (shown in dark grey) and an outside steel ring used for holding the cylinder in place in the stainless-steel housing. This is only one possible configuration of how the novel material can be used.
  • Fig. IB shows the printed porous structure still on the printing support before insertion into the steel housing.
  • Fig. 2A shows an example of 20 pm feature size in the printing procedure.
  • the dashed square is the repeating unit in which the monolith is built upon.
  • Fig. 2B depicts a microscopic picture showing the overview of the structure of 20 pm feature size.
  • Fig. 2C depicts a zoom in Fig. 2B.
  • Fig. 3 depicts pressure drop of the 20 pm pore size printed chromatography in theory (left side) and in the experimental validation (right side).
  • FIG. 4 depicts protein pulse injections at non-binding conditions at different flow rates.
  • Fig. 5 depicts the overlaps between the signal obtained with the H PLC system alone (full black line) and the H P LC system plus the monolithic column (dashed and doted lines). Flow rate starting at 0.25 mL/min at A, to 2.5 mL/min at J, with 0,25 mL/min increments in between.
  • Fig. 6 depicts chromatograms from protein pulse injection on the monolith. Top BSA and bottom lysozyme.
  • Fig. 7 shows typical base materials for monoliths generated by phase separation.
  • 1 depicts methacrylate (2-methylprop-2-enoic acid)
  • 2 depicts glycidyl methacrylate ((oxiran-2-yl)methyl 2- methyl prop-2-enoate)
  • 3 depicts methyl methacrylate (methyl 2- methyl prop-2-enoate)
  • 4 depicts poly(methyl methacrylate).
  • a material with strictly regular channels is provided for separation of biomolecules such as proteins and other biopolymers including bio-nanoparticles, , such as viruses, virus like particles, or exosomes.
  • chromatographic bed material refers to the stationary solid phase used in chromatography.
  • the stationary phase according to the invention exhibits strictly regular channels avoiding wall effects.
  • the stationary phase material may be produced from silicate-based materials, inorganic monolithic materials, and organic polymers, such as vinyl esters, methacrylates, polystyrenes, ethylene glycols, and acrylonitrile- butadiene -styrene or other polymers.
  • the chromatographic bed according to the invention constitutes a stationary phase with strictly regular channels.
  • This strictly regular channels provides for a uniform structure throughout the entire chromatographic bed without any interspaces between the channels.
  • This uniform structure ensures a seamless transition from the enclosure of the chromatographic bed to the center of the chromatographic bed material.
  • the bed material has the same regular structure throughout the whole diameter of the chromatographic bed thereby abolishing the wall effect present in common packed chromatographic columns.
  • a chromatography bed material with straight completely regular channels. These straight and independent channels go from the top to the bottom of the monolithic structure.
  • the wall thickness and the thickness of the pores may be in the same range, or differ in size.
  • the channels may be arranged in parallel.
  • This parallel feature is suggested as the best structure because it is the one with (i) the lowest flow resistance, (ii) the most homogeneous flow field and (iii) the narrowest distribution of mass- transfer distances.
  • it is seen to lack a critical feature as it has no channel interconnectivity. But these interconnectivity could be omitted if the channel width could be guaranteed for all the channels and through the entire length.
  • a chromatography bed material without any gap between the wall and the bed material.
  • the strictly regular channels may have a width in the range of about 0.1 to 100 pm, or in the range of about 0.2 to 50 pm, or in the range of about 0.5 to 25 pm, or in the range of 1 to 10 pm. Therefore, a substantial surface is provided enabling high binding capacity or in case of analytical separation a high peak capacity.
  • the material also abolishes the wall effect of a commonly packed chromatographic column, of a monolith or a stacked membrane. Due to the strictly regular straight and not interconnected channels the chromatographic bed has a homogenous structure. That means that the structure close to the wall and in the center of the bed is identical. This novel arrangement provides for controlling of band spreading is entirely by extra column band broadening as theoretically expected from such a regular structure. A strict control of the internal morphology and regularity of the porous bed (monolith) is made available.
  • the bed for chromatography is achieved by means of two-photon laser polymerization of a polymeric material to form a three-dimensional bed material, wherein: said three-dimensional bed material comprises strictly regular straight channels.
  • the channels may further possess functionalized groups for bioseparation. If necessary, the channels may be functionalized such that the channels carry a functional group adapted for binding to a target molecule. Exemplary binding that can occur between such functional groups and a target molecule, such as a bioparticle or a bionanoparticle, can include ionic bonds, hydrogen bonds, and van der Waals forces. Exemplary functional groups include amine groups (including primary, secondary, tertiary or quaternary amines), sulfonic acid groups, carboxylic acid groups, phosphate groups, and the like including complex structures used for multimodal chromatography including one or more of the aforementioned functional groups, or covalently bound polymers or proteins.
  • the derivatizing reactions to attach such functional groups typically involve reacting a reactive group on the polymer with a molecule containing the desired functional group.
  • a monomer carrying the functional group can be included during the polymerization by a 3D printer.
  • the channel surface can be functionalized with all functional groups used in bioseparation. Specifically groups used in ion-exchange chromatography, hydrophobic interaction chromatography, reversed phase chromatography, normal phase chromatography, multimodal chromatography and affinity chromatography are particularly used.
  • Bioparticles span a large size range comprising, for example, several orders of magnitude from a few hundred nanometers to several tens of micrometers (m m), e.g., exosomes comprise about 50-100 nm sized entities.
  • examples of bioparticles include cells (including bacteria and other microbes), viruses, fungi, spores, virus like particles, extra cellular particles, exosomes of other extracellular vesicles, polymer particles with or without being covered by proteins or nucleic acids or inorganic particles with or without being covered by proteins or nucleic acids.
  • the chromatographic bed may be produced using the technique known as two-photon laser polymerization (2PP). It is carried out via a laser source with a wavelength in the near infrared (NIR) focused in a photosensitive material constituted by a pre-polymer (i.e., monomers and oligomers) and by a photo initiator. This phenomenon results in an overall polymerization of the focal volume, causing in situ cross-linking of the monomers and of the oligomers.
  • the likelihood of two-photon absorption has a quadratic dependence upon the intensity of the IR incident laser beam; consequently, polymerization occurs only within a volume restricted around the focus. Absorption of two independent and simultaneous photons causes an energy transition that cannot be obtained by absorption of a single infrared photon.
  • the method comprises the steps of providing a polymeric resin mixture for polymerization, utilizing two photon polymerization for generating a chromatographic bed material with strictly regular straight or interconnected channels with a seamless transition to the wall structure.
  • the wall of the chromatographic bed can be made of the same material as the wall of the channels.
  • the wall of the regular and /or interconnected channels also represent the wall of the chromatographic bed.
  • no additional housing or coating of the chromatographic bed is needed.
  • the chromatographic bed is comprised in a housing.
  • the housing may be made of glass, stainless steel, or the like.
  • the novel chromatographic bed may be a stand-alone device, e.g., no housing is needed for the chromatographic bed.
  • the chromatographic bed is made as one unit. That means that the chromatographic bed is casted in one piece and ready for use. No packing of a chromatographic column is required.
  • these regular chromatographic beds can be also stacked in order to perform different chromatography modes in a single column. This allows the combination of different separation modes in one single column. This can be used to combine different functionalities, like cation exchange, anion exchange, hydrophobic interaction, hydrophilic interaction, metal-chelate chromatography, affinity chromatography, multimodal chromatography, size exclusion chromatography or any other chromatography mode functionality. Examples
  • the chromatography bed was printed using NanoOne instrument by Upnano with the proprietary material Up-Photo consisting of polymerizable acrylates.
  • the internal structure of the porous part of the monolith was printed with either 20 pm feature size, 10 pm feature size or 5 pm feature size (Figure 2) where the feature size denotes the pore size of the rectangular channel as well as the wall thickness. Microscopic images show the strict regularity of the porous structure and the involved size of wall thickness and pore size (the used feature size).
  • the resulting structures were tested for their solvent combability and hydrodynamic permeability.
  • solvent combability the material shows swelling due to ethyl- alcohol and closing of the pores, but no swelling and no detrimental effects for isopropyl- alcohol or salt buffers of various salt concentrations.
  • hydrodynamic permeability was measured by measuring the pressure drop across the printed material with flow rates ranging from 0.25 to 2.5 mL/min and compared them to the theoretical pressure drop expected for completely open pores of the theoretical dimensions.
  • Pressure drop experiments were performed with high quality water that was previously filtered through 0.22 m m filters (Merck KGaA) and degassed.
  • the HPLC column oven was kept at a constant temperature of 23 C.
  • Pressure drop experiments with an empty monolith housing was performed and later subtracted. The results were evaluated and quantified with the ChromeleonTM 7 software (Thermo Fisher Scientific).
  • Mobile phase A was a 50 mM phosphate buffer, pH 7.0.
  • Mobile phase B was a 50 mM phosphate buffer with 1 M NaCI, pH 7.0.
  • All buffers were filtered through 0.22 m m filters (Merck KGaA) and degassed. Protein solution were prepared in mobile phase A and were filtered with a 0.22 m m syringe filters (Merck KGaA). The system was run at a flow rate of 1 mL.min - 1 and the column oven was kept a constant temperature of 23 C. 20 m L of the sample was loaded.
  • the monolith was equilibrated with 10 column volumes of mobile phase A, eluted with a step gradient with 20 column volumes of 100% mobile phase B, and re - equilibrated with 20 column volumes of mobile phase A.
  • the absorbance at 280 nm was measured.
  • the results were evaluated and quantified with the ChromeleonTM 7 software (Thermo Fisher Scientific).
  • the band broadening is typically quantified in the form of peak variances in individual parts of the system, which are assumed to contribute additively to the total-system peak variances. [0065] In order to gain more information about the channel polydispersity the peak variances from elution peaks were studied.
  • Band deformation is characteristic for laminar flow regimes. Usually, these deformations happen when the residence time is not sufficient for the eluting species to reach uniformity of radial velocity distribution.
  • the printed material may be either directly printed with additional functionalities by adding copolymerizing the functional groups with the acrylic mixture generating the functionality right from the printed material, or the material may be functionalized by the addition of a general reactive group (like glycidyl methacrylate) making all procedures available for additional functionalities that are already developed for the functionalization of monoliths generated by phase separation.
  • the polymerization mixture for the two-photon polymerization may include a certain percentage of attachment points by the addition of glycidyl methacrylate, which is the most commonly used functionalization base for monoliths generated by phase separation.
  • the addition of glycidyl methacrylate enables the functionalization through the present epoxy group with a wide variety of possible reactions to generate positively charged, negatively charged, nonpolar and also complex ligands through the addition of a further NH 2 linkage.

Abstract

La présente invention concerne un système chromatographique comprenant un lit poreux ayant une structure uniforme s'étendant sur le lit chromatographique entier jusqu'à la paroi latérale et son utilisation.
PCT/EP2021/082999 2020-11-25 2021-11-25 Nouveau lit de chromatographie WO2022112416A1 (fr)

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