Automated sample preparation system and methods

Abstract

The invention provides a magnetic beads-based method for automated, accurate and efficient purification and preparation of biological samples for analysis LC-MS analysis.

Description

Patent Application Atty. Docket No. CUR-006PCTAUTOMATED SAMPLE PREPARATION SYSTEM AND METHODSPriority Claims and Related Applications

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 734,222, filed December 16, 2024, the entire content of which is incorporated herein by reference for all purposes.Technical Field of the Invention

[0002] The invention generally relates to a novel method for purification or preparation of biological samples. More particularly, the invention relates to a novel method for efficient, accurate and automated purification and preparation of biological samples for analysis (e.g., LC- MS analysis).Background of the Invention

[0003] The past decade has witnessed a rapidly increasing and evolving need for testing and diagnosis of Drugs-of-Abuse (DOA) and infectious diseases. Clinical and laboratory operators continue to endure heavy burdens resulting in undesirable delays in sample preparation and unacceptable decrease in accuracy of the analytic process.

[0004] Solid phase extraction (SPE) is often used for DOA in oral fluid. SPE uses solid sorbents filled in cartridges or other container formats, such as disks, chromatographic columns, pipette tips and multi-well plates, to selectively capture specific analytes from sample solutions. A standard SPE workflow involves conditioning, sample loading, washing, and elution steps, with the assistance of vacuum or positive pressure to help solvent through the cartridge. The process is time-consuming and challenging to automate.

[0005] Therefore, an urgent need remains for novel and improved sample extraction and purification method that can work with diverse sample types and analytes of interest in an accurate and high throughput fashion.Summary of the InventionPatent ApplicationAttv. Docket No. CUR-006PCT

[0006] The invention relates to an innovative, magnetic beads-based method and system for automated, accurate and efficient extraction and purification of biological samples for analysis (e.g., LC-MS analysis). A key benefit of the disclosed method stems from the incorporation of magnetic beads in the extraction process, which enables the process to isolate and purify the analytes of interest from diverse types of crude samples in a highly efficient and automated manner.

[0007] In one aspect, the invention generally relates to a method for purifying a biological sample. The method comprises: (a) pretreating a sample, wherein the sample is selected from an oral fluid sample or a blood, plasma or serum sample, with a pretreatment buffer; (b) transferring a plurality of magnetic beads to the pretreated sample to absorb analytes of interest; (c) rinsing the plurality of magnetic beads with a wash buffer; and (d) eluting the rinsed magnetic beads with an eluent buffer to collect an eluate comprising one or more analytes of interest.

[0008] In another aspect, the invention generally relates to a method for automated purification of biological samples. The method comprises automatically purifying two or more biological samples in each instance.

[0009] In yet another aspect, the invention generally relates to a composition comprising a plurality of magnetic beads bound thereto one or more analytes of interest.Brief Description of the Drawings

[0010] FIG. 1 shows an exemplary workflow for purifying an oral fluid sample.Definitions

[0011] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 6 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5 or 6.

[0012] In this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference, unless the context clearly dictates otherwise.

[0013] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,Patent ApplicationAttv. Docket No. CUR-006PCT2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.

[0014] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive.

[0015] Any compositions or methods disclosed herein can be combined with one or more of any of the other compositions and methods provided herein.

[0016] The term “comprising”, when used to define compositions and methods, is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. The term “consisting essentially of’, when used to define compositions and methods, shall mean that the compositions and methods include the recited elements and exclude other elements of any essential significance to the compositions and methods. For example, “consisting essentially of’ refers to administration of the pharmacologically active agents expressly recited and excludes pharmacologically active agents not expressly recited. The term consisting essentially of does not exclude pharmacologically inactive or inert agents, e.g., pharmaceutically acceptable excipients, carriers or diluents. The term “consisting of’, when used to define compositions and methods, shall mean excluding trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.Detailed Description of the Invention

[0017] The invention provides a magnetic beads-based method and system for purification and preparation of biological samples. More particularly, the novel method allows for automated, accurate and efficient extraction and purification of biological samples for further analysis, for example, by MS or LC / MS.

[0018] The main feature and advantage of the disclosed method is the employment of magnetic beads, which allows scalability and automation and is suited for a wide range of sample types and analytes of interest. Magnetic beads are made of iron oxide particles, such as particles of magnetite (FesC ), with coatings and surface chemistries designed for desired binding properties. Magnetic beads are characterized by their superparamagnetic properties, which enable the beads to display magnetic behavior only in the presence of an external magnetic field. The sizes and coatings of the beads can be designed according to the types of samples andPatent ApplicationAttv. Docket No. CUR-006PCT analytes of interest. (Farahmandi, et al. Anal. Chim. Acta. 2021 Dec 15, 1188:339183, doi: 10.1016 / j.aca.2021.339183; Zhang, et a . Anal. Biochem. 2019 Aug 15, 579:9-17, doi: 10.1016 / j.ab.2019.05.004; Archer, et al. Anal. Biochem. 2006 Aug 15, 355(2):285-97, doi: 10.1016 / j.ab.2006.05.005; Tian, et al. Foods 2022 Dec 7, 11(24):3944; doi:10.3390 / foods 11243944.)

[0019] In one aspect, the invention generally relates to a method for purifying a biological sample. The method comprises: (a) pretreating a sample, wherein the sample is selected from an oral fluid sample or a blood, plasma or serum sample, with a pretreatment buffer (b) transferring the plurality of magnetic beads to the pretreated sample to absorb analytes of interest; (c) rinsing the contacted magnetic beads with a wash buffer; and (d) eluting the rinsed magnetic beads with an eluent buffer to collect an eluate comprising one or more analytes of interest.

[0020] In certain embodiments, the method further comprises: (e) removing the one or more solvents from the eluate to obtain a purified sample.

[0021] In certain embodiments, the method further comprises: (f) reconstitute the purified sample with a reconstitution buffer.

[0022] In certain embodiments, the method further comprises: (g) analyzing the reconstituted sample for the one or more analytes.

[0023] Any suitable magnetic beads may be used in the methods of the invention.

[0024] In certain embodiments, the magnetic beads are characterized by one or more of the following characteristics: an ion exchange capacity greater than about 400 meq / g; a hydrophobic interaction (e.g., having about 10 to about 30% phenyl or biphenyl functional group); a pore size less than about 120 A; and / or a particle size of about 5 pm to about 30 pm.

[0025] In certain embodiments, the pretreatment buffer comprises one or more weak acids (e.g., formic acid or acetic acid).

[0026] In certain embodiments, the conditioning buffer comprises pure methanol or acetonitrile.

[0027] In certain embodiments, the wash buffer comprises a first wash buffer comprising water and one or more weak acids (e.g., formic acid) and a second wash buffer with a pH of about 2 to about 4 comprising a low percentage (e.g., about 40% to about 60%) an organic solvent and one or more weak acids (e.g., formic acid).Patent ApplicationAttv. Docket No. CUR-006PCT

[0028] In certain embodiments, the eluent buffer comprises a high percentage (e.g., 95%) of an organic solvent (e.g., acetonitrile) with ammonium hydroxide with a pH of about 10.

[0029] In certain embodiments, the reconstitution buffer comprises: about 10% to about 20% methanol in water.

[0030] In certain embodiments, the reconstituted sample is analyzed qualitatively or quantitatively for one or more analytes by liquid chromatography-mass spectrometry (LC-MS) analysis.

[0031] Any suitable samples may be purified according to the methods disclosed herein.

[0032] In certain embodiments, the sample to be purified is comprises an oral fluid (e.g., oral fluid or spit). For oral fluid samples, certain components (e.g., proteins, insoluble particles and surfactants) if present are preferably removed from the collection device or container.

[0033] In certain embodiments, the sample to be purified comprises a blood material (e.g., blood, plasma or serum).

[0034] In certain embodiments, the sample is whole blood.

[0035] For oral fluid samples, certain components (e.g., proteins, lipids, insoluble particles) if present are preferably removed from the collection device or container.

[0036] In certain embodiments, the sample to be purified is substantially free of insoluble particles.

[0037] In certain embodiments, the sample to be purified is substantially free of surfactants.

[0038] In certain embodiments, the sample to be purified is substantially free of proteins and lipids.

[0039] The disclosed methods are suitable for purifying samples to be further analyzed for one or more analytes of interest.

[0040] In certain embodiments, the one or more analytes of interest comprise one or more exogenous compounds, for examples, drugs of abuse and therapeutical agents.

[0041] Exemplary drugs of abuse include marijuana (e.g., tetrahydrocannabinol (THC)), opiates (e.g., morphine and oxycodone), and benzodiazepines (e.g., alprazolam).

[0042] Exemplary therapeutical agents include cyclosporine.

[0043] In certain embodiments, the one or more analytes of interest comprise one or more endogenous compounds, for examples, steroid hormones and biogenic amines.Patent Application Atty. Docket No. CUR-006PCT

[0044] In certain embodiments, the one or more analytes of interest comprise one or more immunosuppressants.

[0045] Exemplary steroid hormones include estrone.

[0046] Exemplary biogenic amines include histamine.

[0047] In certain embodiments, the analytes of interest for a sample to be purified comprise10 or more exogenous compounds and / or endogenous compounds.

[0048] In certain embodiments, the analytes of interest for a sample to be purified comprise50 or more exogenous compounds and / or endogenous compounds.

[0049] Non-limiting examples of analytes of interest can be found in Table 1.Table 1. Exemplary AnalytesPatent ApplicationAttv. Docket No. CUR-006PCT

[0050] In another aspect, the invention generally relates to a method for automated extraction or purification of biological samples. The method comprises automatically extracting or purifying two or more biological samples in each instance.

[0051] In certain embodiments, ten or more biological samples are processed in each instance.

[0052] In yet another aspect, the invention generally relates to a composition comprising a plurality of magnetic beads bound thereto one or more analytes of interest (e.g., selected from Table 2).

[0053] The following examples are meant to be illustrative of the practice of the invention, and not limiting in any way.ExamplesGeneral Steps in Sample Extraction and Purification

[0054] Sample pretreatment: Mix specimen with suitable buffer to adjust pH of the sample. For quantitative testing, internal standard is usually added during this step.

[0055] Magnetic beads activation (optional): Mix magnetic beads (MB) with organic solvent to wet and activate magnetic beads if needed.

[0056] Sample loading: MB is collected from activation buffer and mix with pretreated sample.

[0057] Rinse: MB is collected from sample solution and mixed with wash buffer to remove interfering substances. Wash buffer normally contains 0-50% organic solvent and low concentration of acid to keep the pH below 5.

[0058] Elute: MB is collected from wash buffer and mixed with elute buffer to elute analyte of interest. Elution buffer usually consists of high concentration of organic solvent and pH modifier.Exemplary ProceduresPatent Application Atty. Docket No. CUR-006PCT

[0059] An exemplary procedure is provided below.Prepare condition plate by transferring 1 mL methanol into each well of 96 deep well plate (DWP)Prepare two wash plate by transferring 1 mL 40% methanol to the platesPrepare elution plate by transferring 1 mL of 95% acetonitrile and 5% ammonium hydroxide to the platePrepare beads plate by transferring 10 mg magnetic beads to each well of 96 deep well plate (DWP)Prepare sample plate by transferring 0.5 mL of specimen to the plate, then dispense 0.5 mL 4% formic acid to each wellCollect the magnetic beads and transfer them to condition plate, Mix beads for 1 min.Collect beads and transfer to sample plate, mix for 1 min.Collect beads and transfer to wash plate, mix for 1 min.Repeat the wash step.Collect beads from wash plate and transfer to elution plate, mix for 1 min.Collect beads from elution plate.Place elution plate in nitrogen evaporator until all wells are dryAdd 0.5 mL 10% methanol to each well and seal the plateThe plate is ready for LC-MS analysisRecovery Rate of Exemplary Analytes:Table 2. Exemplary Recovery RatesPatent ApplicationAttv. Docket No. CUR-006PCTPatent ApplicationAtty. Docket No. CUR-006PCTExtraction of Immunosuppressants

[0060] This application note describes the extraction of sirolimus, tacrolimus, everolimus, and cyclosporin A from whole blood samples using magnetic beads-based solid phase extraction (mSPE) prior to LC-MS / MS analysis. These four drugs exhibit strong immunosuppressive properties and are commonly used to prevent organ transplant rejection and manage autoimmune diseases. Due to the associated risk of immunodeficiency, patients undergoing treatment require continuous therapeutic drug monitoring (TDM), which depends on reliable and robust analytical methods for accurate drug quantification.

[0061] Traditionally, immunoassays have been employed for this purpose; however, they are costly, time-consuming, and prone to cross-reactivity issues. In contrast, LC-MS / MS assays are gaining popularity due to their superior sensitivity and selectivity. Most current LC-MS methodologies rely on protein precipitation for sample extraction — a process that is labor- intensive, time-consuming, and susceptible to ion suppression. While solid-phase extraction (SPE) can be used to further clean protein-precipitated samples, it significantly increases both cost and processing time.

[0062] Here is an example of a streamlined, automated sample preparation method using magnetic beads-based system disclosed herein, which effectively minimizes matrix effects and enhances assay performance.

[0063] The magnetic beads-based system integrates blood cell lysis and analyte absorption into one step. The whole SPE extraction process can be easily automated on most magnetic bead processor, such as Thermo KingFisher Flex.

[0064] Analytes: Sirolimus, Tacrolimus, Everolimus, Cyclosporin A.

[0065] Equipment and Supplies: Thermo KingFisher Flex, CURA mSPE plate, KingFisher Flex tip comb, KingFisher 96 deep well plate, HPLC, LCMS.

[0066] Recovery Rate and Matrix Effect:

[0067] Extraction efficiencies and matrix effects were evaluated for each analyte at the following concentrations: Sirolimus (500 ng / mL), Tacrolimus (20 ng / mL), Everolimus (250Patent ApplicationAtty. Docket No. CUR-006PCT ng / mL), and Cyclosporin (500 ng / mL), following the method described by Matuszewski et al. (n = 4 per analyte set) [https: / / pubs.acs.org / doi / 10.1021 / ac020361s],

[0068] Three experimental sets were prepared:• Set A: Drug standards were added to drug-free whole blood from healthy donors before mSPE, and internal standards (IS) were added after mSPE.• Set B: Drug-free whole blood was fortified with both drug standards and IS after mSPE.• Set C: Water was fortified with both drug standards and IS after mSPE.

[0069] Extraction efficiency (%) was calculated as:(Mean peak area ratio of Set A / Mean peak area ratio of Set B)x 100

[0070] Matrix effect (%) was calculated as:(Mean peak area ratio of Set B / Mean peak area ratio of Set C)* 1001. Recovery Rate (Set A / Set B x 100%)2. Matrix Effect (Set B / Set C x 100%)3. Matrix Effect by IS ResponseSet B IS ResponsePatent ApplicationAtty. Docket No. CUR-006PCTSet C IS ResponseMatrix Effect by IS Response

[0071] Evaluation of Variation of Analyte and IS response:

[0072] Negative whole blood collected from healthy donors was fortified with both drug standard and internal standard solutions prior to mSPE sample extraction. Four replicates were analyzed to evaluate the variation of analyte and IS response (Peak Area). The CV of the response of all analytes were below 8%.

[0073] Applicant’s disclosure is described herein in preferred embodiments with reference to the Figures, in which like numbers represent the same or similar elements. Reference throughoutPatent ApplicationAttv. Docket No. CUR-006PCT this specification to “one embodiment,” “an embodiment,” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0074] The described features, structures, or characteristics of Applicant’s disclosure may be combined in any suitable manner in one or more embodiments. In the description, herein, numerous specific details are recited to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that Applicant’s composition and / or method may be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the disclosure.

[0075] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Methods recited herein may be carried out in any order that is logically possible, in addition to a particular order disclosed.Incorporation by Reference

[0076] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made in this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure.EquivalentsPatent Application Atty. Docket No. CUR-006PCT

[0077] The representative examples are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples and the references to the scientific and patent literature included herein. The examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

Patent ApplicationAttv. Docket No. CUR-006PCTWhat is Claimed is:CLAIMS1. A method for purifying a biological sample, comprising:(a) pretreating a sample, wherein the sample is selected from an oral fluid sample or a blood, plasma or serum sample, with a pretreatment buffer;(b) transferring a plurality of magnetic beads to the pretreated sample to absorb analytes of interest;(c) rinsing the contacted magnetic beads with a wash buffer; and(d) eluting the plurality of magnetic beads with an eluent buffer to collect an eluate comprising one or more analytes of interest.

2. The method of claim 1, further comprising:(e) removing the one or more solvents from the eluate to obtain a purified sample.

3. The method of claim 2, further comprising:(f) reconstitute the purified sample with a reconstitution buffer.

4. The method of any one of claims 1-3, further comprising:(g) analyzing the reconstituted sample for the one or more analytes.

5. The method of any one of claims 1-4, wherein the plurality of magnetic beads is characterized by having: an ion exchange capacity greater than about 400 meq / g; a hydrophobic interaction by adding 10-30% phenyl or biphenylfunctional group; a pore size less than about 120 A; and / or a particle size of about 5 pm to about 30 pm.

6. The method of any one of claims 1-5, wherein the pretreatment buffer comprises weak acid; the conditioning buffer comprises: pure methanol;Patent ApplicationAttv. Docket No. CUR-006PCT the wash buffer comprises a first wash buffer comprising water and weak acid and a second wash buffer comprising low percentage acidified organic solvent with a pH of about 2 to about 4; the eluent buffer comprises: high percentage organic solvent with ammonium hydroxide with a pH of about 10; and / or the reconstitution buffer comprises: about 10% to about 20% methanol in water.

7. The method of any one of claims 4-6, wherein the analysis comprises performing liquid chromatography-mass spectrometry (LC-MS) analysis.

8. The method of any one of claims 4-7, wherein the analysis is qualitatively.

9. The method of any one of claims 4-7, wherein the analysis is quantitatively.

10. The method of any one of claims 1-9, wherein the sample comprises an oral fluid.

11. The method of any one of claims 1-9, wherein the sample comprises a blood, plasma or serum material.

12. The method of any one of claims 1-9, wherein the sample comprises whole blood.

13. The method of any one of claims 1-12, wherein the sample is substantially free of insoluble particles.

14. The method of any one of claims 1-13, wherein the sample is substantially free of surfactants.

15. The method of any one of claims 1-14, wherein the sample is substantially free of proteins and lipids.Patent ApplicationAttv. Docket No. CUR-006PCT16. The method of any one of claims 1-15, wherein the one or more analytes of interest comprise one or more exogenous compounds.

17. Th method of claim 16, wherein the exogenous compounds are selected from drugs of abuse and therapeutical agents.

18. Th method of claim 17, wherein the drugs of abuse are selected from opiates, marijuana, and benzodiazepines.

19. The method of any one of claims 1-15, wherein the one or more analytes of interest comprise one or more endogenous compounds.

20. Th method of claim 19, wherein the endogenous compounds comprise a compound selected from steroid hormones and biogenic amines.

21. The method of any one of claims 1-15, wherein the one or more analytes of interest comprise one or more immunosuppressants.

22. The method of any one of claims 1-15, wherein the one or more analytes of interest are selected from Table 1.

23. A method for automated purification of biological samples, comprising performing the method of any one of claims 1-22 wherein two or more biological samples are automatically handled in each instance.

24. The method of claim 23, wherein ten or more biological samples are handled in each instance.

25. The method of claim 23 or 24, wherein the analytes of interest comprise 10 or more exogenous compounds and / or endogenous compounds.Patent ApplicationAttv. Docket No. CUR-006PCT26. The method of claim 23 or 24, wherein the analytes of interest comprise 50 or more exogenous compounds and / or endogenous compounds.

27. A composition comprising a plurality of magnetic beads bound thereto one or more analytes of interest selected from Table 1.

28. The composition of claim 27, wherein the magnetic beads are characterized by: a pore size less than about 120 A; and / or a particle size of about 5 pm to about 30 pm.

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