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EP-4443155-B1 - ENCAPSULATED PRE-ANALYTIC WORKFLOWS FOR FLOW-THROUGH DEVICES, LIQUID CHROMATOGRAPHY AND MASS SPECTROMETRIC ANALYSIS

EP4443155B1EP 4443155 B1EP4443155 B1EP 4443155B1EP-4443155-B1

Inventors

  • WYNDHAM, Kevin Daniel
  • BROUSMICHE, DARRYL W.

Dates

Publication Date
20260513
Application Date
20170728

Claims (15)

  1. An encapsulated workflow reagent (5) comprising an encapsulating material and a workflow reagent encapsulated within the encapsulating material, wherein the encapsulating material is attached to a surface of a scaffolding material (B), wherein the scaffolding material is a non-porous solid, wherein the workflow reagent is an enzyme, a surfactant, a labeling reagent, a reactive compound, an internal standard, an external standard or a combination thereof, and wherein the encapsulating material is made with one or more polymers configured to provide a controlled release of the workflow reagent.
  2. The encapsulated workflow reagent according to Claim 1, wherein the workflow reagent is an enzyme.
  3. The encapsulated workflow reagent according to Claim 1, wherein the encapsulated workflow reagent comprises a material having the formula [(B)-(Y) n )] o -EM (Formula I), where EM represents the encapsulating material; B represents the scaffolding material; Y is a linker group between the encapsulating material and the surface of the scaffolding material; o is an integer greater than 0; and n is an integer greater than or equal to 1.
  4. The encapsulated workflow reagent according to Claim 3, wherein the linker group is of the formula represented by Formula II wherein n 1 is an integer from 0-30; n 2 is an integer from 0-30; each occurrence of R 1 , R 2 , R 3 and R 4 independently represents hydrogen, fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, a protected or deprotected alcohol, a zwiterion, or a group Z ; Z represents: a) a surface attachment group having Formula III: (B 1 ) x (R 5 ) y (R 6 ) z Si- Formula III: wherein x is an integer from 1-3, y is an integer from 0-2, z is an integer from 0-2, and x+y+z = 3 each occurrence of R 5 and R 6 independently represents methyl, ethyl, n -butyl, iso -butyl, tert- butyl, iso -propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwiterion group; and B 1 represents -OR 7 , -NR 7' R 7" , -OSO 2 CF 3 , or -Cl; where each of R 7, R 7' and R 7" represents hydrogen, methyl, ethyl, n -butyl, iso -butyl, tert- butyl, iso -propyl, thexyl, phenyl, branched alkyl or lower alkyl; b) an attachment to a surface group through a direct carbon-carbon bond formation or through a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or urethane linkage; or c) an adsorbed, surface group that is not covalently attached to the surface of the material; Y' represents a direct bond; a heteroatom linkage; an ester linkage; an ether linkage; an thioether linkage; an amine linkage; an amide linkage; an imide linkage; a urea linkage; a thiourea linkage; a carbonate linkage; a carbamate linkage; a heterocycle linkage; a triazole linkage; a urethane linkage; a diol linkage; a polyol linkage; an oligomer of styrene, ethylene glycol, or propylene glycol; a polymer of styrene, ethylene glycol, or propylene glycol; a carbohydrate group, a multi-antennary carbohydrates, a dendrimer or dendrigraphs, or a zwitterion group; and A represents attachment to the encapsulating material by a ionic group, non-covalently attachment group or by a direct bond including (but not limited to): a heteroatom linkage; an ester linkage; an ether linkage; an thioether linkage; an amine linkage; an amide linkage; an imide linkage; a urea linkage; a thiourea linkage; a carbonate linkage; a carbamate linkage; a heterocycle linkage; a triazole linkage; a urethane linkage; a diol linkage; a polyol linkage; an oligomer of styrene, ethylene glycol, or propylene glycol; a polymer of styrene, ethylene glycol, or propylene glycol; a carbohydrate group, a multi-antennary carbohydrates, a dendrimer or dendrigraphs, or a zwitterion group.
  5. The encapsulated workflow reagent according to Claim 1, wherein the workflow reagent is released over a period of time.
  6. The encapsulated workflow reagent according to Claim 1, wherein the encapsulating material comprises a primary encapsulation shell that further encapsulates one or more additional encapsulation shells comprising one or more additional workflow reagents.
  7. The encapsulated workflow reagent according to Claim 6, wherein the one or more additional encapsulation shells are in the form of microcapsules separately contained within the primary encapsulation shell.
  8. The encapsulated workflow reagent according to Claim 7, wherein the one or more additional workflow reagents are released at the same time or wherein the one or more additional workflow reagents are released sequentially.
  9. The encapsulated workflow reagent according to Claim 6, wherein the one or more additional encapsulation shells are in the form of concentric encapsulation shells such that the workflow reagents are sequentially released.
  10. A sample preparation device comprising an encapsulated workflow reagent according to Claim 1.
  11. The sample preparation device according to Claim 10, wherein the device is selected from the group consisting of chromatographic columns, thin layer plates, filtration membranes, sample cleanup devices and microtiter plates; packings for HPLC columns; solid phase extraction (SPE); ion-exchange chromatography; magnetic bead applications; affinity chromatographic and SPE sorbents; sequestering reagents; solid supports for combinatorial chemistry; solid supports for oligosaccharide, polypeptides, and/or oligonucleotide synthesis; solid supported biological assays; capillary biological assay devices for mass spectrometry, templates for controlled large pore polymer films; capillary chromatography; electrokinetic pump packing materials; packing materials for microfluidic devices; polymer additives; catalysis supports; and packings materials for microchip separation devices.
  12. A method for a method for preparing a sample for analysis comprising the steps of: providing a sample preparation device comprising an encapsulated workflow reagent according to Claim 1; introducing a sample to the sample preparation material; and allowing the sample to remain with the sample preparation material for sufficient time to release the encapsulated workflow reagent.
  13. The method for preparing a sample for analysis according to Claim 12 further comprising adding a pore forming agent to induce release of the encapsulated workflow reagent.
  14. The encapsulated workflow reagent according to Claim 1, wherein the scaffolding material is in the form of particles having an average particle size of about 10-70 µm.
  15. The encapsulated workflow reagent according to Claim 2, wherein the enzyme is a protease, cellulase, lipase, amylase, glucoamylase, glucose isomerase, xylanase, phytase, arabinanase, polygalacturonase, hydrolase, chymosin, urease, pectinase, beta-gluconase, ligase, glycosidase, polymerase, phosphatase, kinase, exopeptidase, endopeptidase, aminopeptidase, or eramidase, optionally wherein the enzyme is selected from trypsin, PNGase F, PNGase A, pepsin, chymotrypsin, peptidase, bromelain, papain, IdeS, IdeZ, elastase, carboxypeptidase A, capthepsin D, capthepsin E or mixtures thereof.

Description

THIS APPLICATION CLAIMS PRIORITY TO U.S. PROVISIONAL PATENT APPLICATION SERIAL NO. 62/367,948, FILED JULY 28, 2016, ENTITLED ENCAPSULATED PRE-ANALYTIC WORKFLOWS FOR FLOW-THROUGH DEVICES, LIQUID CHROMATOGRAPHY AND MASS SPECTROMETRIC ANALYSIS. BACKGROUND OF THE INVENTION Liquid chromatography and mass spectrometry are essential tools in quantifying, analyzing and characterizing a wide variety of molecules. Complex molecules, such as biomolecules, require multiple processing steps prior to their introduction to flow through devices for analysis. Low molecular weight substances in biological material and bodily fluids require similarly time-consuming, costly and labor-intensive sample-pretreatment steps. Without such preparation, direct injection of protein-containing samples would result in improper and incomplete analysis as well as the accumulation of unwanted species on the chromatographic support materials thereby irreversibly damaging the chromatography column. Traditional sample preparation often requires multiple steps of reaction and separation. For example, for a particular protein sample to be completely analyzed for a particular characteristic, it may be necessary to digest the sample with a particular enzyme or surfactant, or both. The digested peptides may then need to be labeled in some way to allow for proper detection. In each of these steps, samples are allowed to react with the particular reagent and then must be separated to remove unwanted materials and reagents prior to chromatographic or spectroscopic analysis. As an example, a protein digest workflow that uses an affinity capture step requires multiple reagent addition steps, vortexing, centrifuging and heating steps. In a manual mode this requires the preparation of reagents, multiple additions and liquid transfer steps, movement of plates to and from heating blocks, centrifuges, and vortex mixers. This type of manual workflow can be labor intensive and can result in high variability, difficulty transferring and replicating results. The use of automation can be used to improve reproducibility and reduce user involvement. However, automation requires a significant investment in equipment, supporting infrastructure and requires user generated scripts. As such the use of automation is often only available in high volume testing laboratories. As such, there remains a need for a moderate throughput device that allows for a singular flow-through workflow device which allows for robust and simplified workflows. "Continuous generation of hydrogel beads and encapsulation of biological materials using a microfluidic droplet-merging channel", MICROFLUIDICS AND NANOFLUIDICS, SPRINGER, BERLIN, DE, vol. 5, no. 4, 21 February 2008 (2008-02-21), pages 541-549, discloses an encapsulated workflow reagent comprising an encapsulating material and a workflow reagent encapsulated within the encapsulating material (said reagent inducing e.g. digestion). BRIEF SUMMARY OF THE INVENTION This invention relates to encapsulated reagents for sample and workflow preparation, use thereof, and devices comprising the same. Workflows which can be used in the materials and methods of the invention include, but are not limited to, reduction or alkylation reagent workflows, protein digest workflows, proteolytic enzyme and protease workflows, and workflows for the analysis of amino acids, glycoproteins, pesticides and polar compound analysis. The use of encapsulated reagents for digestion, hydrolysis and denaturation (e.g., trypsin, PNGase F, pepsin, IdeS, IdeZ) improves stability and flowability of these materials. The use of encapsulated reagents for hydrolytically unstable and reactive molecules (e.g., WATERS TECHNOLOGIES CORPORATION ACCQ•FLUOR™ labeling agent, WATERS TECHNOLOGIES CORPORATION RAPIFLUOR-MS™ tagging reagent, acid-labile surfactants, acyl chlorides, chloroformate esters, succinimydl carbonates, esters and isocyanates) allows for improved stability and introduction at specific points within a workflow. The use of encapsulated reagents for reactive agents such as reduction agents and alkylating agents (e.g., dithiothreitol (DTT), iodoacetamide, 2-mercaptoehtanol, 2-mercaptoethylamine HCl, Tris (2-carboxyethyl) phosphine hydrochloride, 4-vinylpyridine, haloacyl reagents (e.g. acetylchloride), N-ethylmaleimide, bromoalkanes, acrylamide, sodium borohydride, sodiumcyanoborohydride and other similar reducing agents (e.g. metal hydrides) allows for similarly improved stability and introduction at specific points within a workflow. Encapsulated reagents can further be used for stabilizing thermally sensitive standards in the absence of a singular workflow device. Encapsulated reagents can further be used for stabilizing workflow reagents containing sulfur or otherwise having a strong odor. The use of encapsulated reagents for such compounds allows for the odor to be masked during sample preparation and analysis while maintaining the efficacy and efficiency of the rea