Pressing and mixing thin film enzyme reactor and preparation method thereof

Abstract

Disclosed herein are methods and devices for accelerating the substrate-enzyme reaction rate of sample preparation in biological treatment assays using compressed and mixed films containing predetermined ratios of biological substrates, such as polypeptides, proteins, nucleic acids, sugars or lipids, and enzymes.

Description

[0001] Cross - Reference to Related Applications

[0002] This patent application is an international application claiming the priority and benefit of U.S. Provisional Application No. 63 / 383,779, filed on November 15, 2022, the content of which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This patent application contains a sequence listing, which has been electronically submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy created on November 9, 2023 is named WAC - 399WO_SL.xml and is 22,038 bytes in size. Background Art

[0005] The time required for preparative reactions in analytical techniques (especially in the bioprocessing space) can severely delay the workflow. These delays impede the acquisition of real - time results, which is an important requirement in feedback - loop regulation of bioreactor design. The acceleration of small - molecule reactions in microdroplets has been demonstrated. There are four hypothesized mechanisms for the acceleration of small - molecule reactions in microdroplets: (1) dipole alignment and partial solvation at the droplet surface; (2) concentration effects due to localization and confinement at the droplet surface; (3) concentration effects due to droplet evaporation; and (4) the influence of charge and electric fields. However, due to the preferential localization of folded proteins inside the droplets, only spatial confinement, evaporation effects, and electric - field effects have been observed for protein - enzyme reactions. Thus, the observed acceleration factors are in the range of 10 3 -10 4 range, rather than the 10 5 -10 6 range reported for some small - molecule reactions. One limiting parameter of microdroplets is the short lifespan inherent to this substance. Thin films have also been used for reaction acceleration and are effective for similar reasons as microdroplets. Although the acceleration factors observed for films are generally lower than those observed for microdroplets, the lifespan of films can be extended by many orders of magnitude compared to microdroplets, thus providing a simple way to overcome the reduction in acceleration factors. The present disclosure provides pressed and mixed thin films and methods for their preparation as a solution for accelerating the reaction rate of substrate - enzyme systems (SES). Summary of the Invention

[0006] In certain embodiments, methods for accelerating the reaction rate of a substrate - enzyme system (SES) are disclosed herein, including preparing a pressed and mixed thin film comprising a predetermined ratio of a substrate (e.g., polypeptide, protein, nucleic acid, sugar, or lipid) and an enzyme. An apparatus for producing the thin film is also disclosed.

[0007] In one aspect, the present disclosure provides a method for preparing a protein sample in a thin film for a biological assay, the method comprising: (a) combining an enzyme and a protein substrate at a predetermined ratio to produce a substrate-enzyme mixture (e.g., SES); (b) depositing the mixture of step (a) in a bottom conical or frustoconical surface; and (c) pressing the mixture between the bottom conical or frustoconical surface and a top conical or frustoconical surface by applying a predetermined force between the top conical or frustoconical surface and the bottom conical or frustoconical surface, wherein the top conical or frustoconical surface and the bottom conical or frustoconical surface are vertically oriented and configured to produce a nested conical or frustoconical interface; thereby forming a thin film containing the substrate-enzyme (e.g., protein-enzyme) mixture in the nested conical interface. In certain embodiments, the predetermined ratio of enzyme to substrate (e.g., protein) is about 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, or 1:200 (moles of enzyme: moles of substrate). In certain embodiments, the substrate-enzyme mixture is present in solution at a concentration of 0.1 mg / mL to 20 mg / mL (e.g., 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, and 20 mg / mL). In certain embodiments, the substrate-enzyme mixture is in a solution of 10 μL to 100 μL (e.g., 10 μL to 100 μL, 15 μL to 95 μL, 20 μL to 90 μL, 25 μL to 85 μL, 30 μL to 80 μL, 35 μL to 75 μL, 40 μL to 70 μL, 45 μL to 65 μL, and 50 μL to 60 μL).In certain embodiments, the solution containing the substrate-enzyme system has a viscosity of 0.1 mPa·s to 2.0 mPa·s (e.g., 0.1 mPa·s to 2.0 mPa·s, 0.2 mPa·s to 1.9 mPa·s, 0.3 mPa·s to 1.8 mPa·s, 0.4 mPa·s to 1.7 mPa·s, 0.5 mPa·s to 1.6 mPa·s, 0.6 mPa·s to 1.5 mPa·s, 0.7 mPa·s to 1.4 mPa·s, 0.8 mPa·s to 1.3 mPa·s, 0.9 mPa·s to 1.2 mPa·s, and 1.0 mPa·s to 1.1 mPa·s). In certain embodiments, the predetermined force is between 3 pounds and 20 pounds (e.g., 3 pounds to 20 pounds, 4 pounds to 19 pounds, 5 pounds to 18 pounds, 6 pounds to 17 pounds, 7 pounds to 16 pounds, 8 pounds to 15 pounds, 9 pounds to 14 pounds, 10 pounds to 13 pounds, and 11 pounds to 12 pounds). In certain embodiments, the predetermined force is between 10 pounds and 15 pounds (e.g., 10 pounds to 15 pounds, 11 pounds to 14 pounds, and 12 pounds to 13 pounds). In certain embodiments, the predetermined force is applied and maintained for a duration between 3 s and 5 s (e.g., 3.0 s to 5.0 s, 3.1 s to 4.9 s, 3.2 s to 4.8 s, 3.3 s to 4.7 s, 3.4 s to 4.6 s, 3.5 s to 4.5 s, 3.6 s to 4.4 s, 3.7 s to 4.3 s, 3.8 s to 4.2 s, and 3.9 s to 4.1 s). In certain embodiments, after the force is applied, the force is withdrawn for a duration between 1 s and 2 s (e.g., 1.0 s to 2.0 s, 1.1 s to 1.9 s, 1.2 s to 1.8 s, 1.3 s to 1.7 s, and 1.4 s to 1.6 s). In certain embodiments, the application and withdrawal of the force are carried out within a total duration of about 30 s. In certain embodiments, compared to the reaction rate between the enzyme and the substrate in the bulk solution, this method increases the reaction rate between the enzyme and the substrate (e.g., protein). In certain embodiments, the increase in the reaction rate is due to the binding rate of the enzyme to the substrate (K. on) increase. In certain embodiments, the enzyme is selected from proteases, glycosylating enzymes, and phosphatases. In certain embodiments, the protease is trypsin, chymotrypsin, immunoglobulin-degrading enzyme (IgDE), immunoglobulin G-degrading enzyme (IdeS) of Streptococcus pyogenes, Asp-N protease, or Lys-C protease. In certain embodiments, the glycosylating enzyme is peptide:N-glycosidase F (PNGase F). In certain embodiments, the phosphatase is alkaline phosphatase (ALP). In certain embodiments, the bioprocessing assay is liquid chromatography (LC), mass spectrometry (MS), or a combination thereof. In certain embodiments, the bioprocessing assay is peptide mapping, peptide monitoring, proteomics, protein quantification, glycan characterization, glycomics, and glycoprotein characterization. In certain embodiments, the thickness of the thin film is not greater than about 100 μm, about 90 μm, about 80 μm, about 70 μm, about 60 μm, about 50 μm, about 40 μm, about 30 μm, about 20 μm, or about 10 μm. In certain embodiments, the thin film has a thickness of not greater than about 50 μm.

[0008] In one aspect, the present disclosure provides an apparatus for producing a thin film (e.g., a thin film including SES), the apparatus comprising: (a) a vertically oriented convex top conical or frustoconical surface, wherein the base of the top conical or frustoconical surface is attached to a top base member; (b) a vertically oriented concave bottom conical or frustoconical surface configured to receive a sample volume, wherein the bottom conical or frustoconical surface is within an interior of a cylindrical bottom base member; (c) a fixed linear actuator configured to move along a vertical axis and operably connected to a raised cylindrical surface on the top base member or operably connected to the bottom base member; (d) a fluid inlet port operably connected to the top conical or frustoconical surface and / or the bottom conical or frustoconical surface; (e) one or more (e.g., 1, 2, 3, 4 or more) heating elements (e.g., cartridge heaters or infrared lasers) operably connected to a temperature controller and a relay circuit; (f) a thermistor configured to be placed within a thermistor bore in the raised cylindrical surface of the top base member, the thermistor being operably connected to the temperature controller; wherein the top conical or frustoconical surface and the bottom conical or frustoconical surface are configured to produce nested conical or frustoconical interfaces. In certain embodiments, the thin film is configured for depositing one or more (e.g., 1, 2 or more) biological agents (e.g., enzymes and enzyme substrates such as proteins, nucleic acids, sugars or lipids) on a surface of the thin film. In certain embodiments, the one or more biological agents are target proteins. In certain embodiments, the target protein is an enzyme. In certain embodiments, the one or more biological agents further include a substrate of the enzyme. In certain embodiments, the linear actuator is configured to produce a predetermined force between 3 pounds and 20 pounds (e.g., 3 pounds to 20 pounds, 4 pounds to 19 pounds, 5 pounds to 18 pounds, 6 pounds to 17 pounds, 7 pounds to 16 pounds, 8 pounds to 15 pounds, 9 pounds to 14 pounds, 10 pounds to 13 pounds, and 11 pounds to 12 pounds) between the top conical or frustoconical surface and the bottom conical or frustoconical surface. In certain embodiments, the linear actuator is configured to produce an angular motion with an offset angle of 10° - 30°. In certain embodiments, each of the top and / or bottom conical or frustoconical surfaces has a slant height of 3 cm to 15 cm (e.g., 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, and 15 cm). In certain embodiments, the bottom conical or frustoconical surface has a vertical height of 2.85 cm to 10 cm (e.g., 2.85 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, and 10 cm).In certain embodiments, the top conical or frustoconical surface and the bottom conical or frustoconical surface each have an apex angle (e.g., an inclination angle) of 45°. In certain embodiments, the top frustoconical surface and the bottom frustoconical surface include an upper base with a diameter of 1 mm to 10 mm (e.g., 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, and 10 cm). In certain embodiments, each of the top conical or frustoconical surface and the bottom conical or frustoconical surface is independently composed of a material selected from stainless steel, poly(methyl acrylate), and glass. In certain embodiments, the fluid inlet port is operably connected to a sample injection device. In certain embodiments, the top conical or frustoconical surface is actuated while the bottom conical or frustoconical surface is stationary. In certain embodiments, the bottom conical or frustoconical surface is actuated while the top conical or frustoconical surface is stationary. In certain embodiments, the bottom conical or frustoconical surface is configured to accommodate a fluid volume between at least 10 μL and 100 μL (e.g., 10 μL to 100 μL, 15 μL to 95 μL, 20 μL to 90 μL, 25 μL to 85 μL, 30 μL to 80 μL, 35 μL to 75 μL, 40 μL to 70 μL, 45 μL to 65 μL, and 50 μL to 60 μL). In certain embodiments, the thickness of the thin film is not greater than about 100 μm, about 90 μm, about 80 μm, about 70 μm, about 60 μm, about 50 μm, about 40 μm, about 30 μm, about 20 μm, or about 10 μm. In certain embodiments, the thin film has a thickness of not greater than about 50 μm.

[0009] Definition

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In general, the nomenclature and techniques used in connection with immunology, oncology, cell and tissue culture, molecular biology, and the chemistry and hybridization of proteins and oligonucleotides or polynucleotides described herein are those well known and commonly used in the art. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and do not limit any subject matter claimed. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0011] As used herein, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "a protein" includes a single protein or multiple proteins.

[0012] As used herein, unless the context clearly indicates otherwise, all numerical values or ranges of numerical values include all integers within such ranges or covering such ranges, and fractions of values or integers within or covering the ranges. Thus, for example, reference to a range of 90%-100% includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so on. As another example, reference to a range of 1-fold - 5,000-fold includes 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, etc., as well as 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, etc., 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, etc., and so on.

[0013] As used herein, the term "about" a number means including the number and a range that is 10% below the number to 10% above the number. The term "about" a range means 10% below the lower limit of the range and spanning to 10% above the upper limit of the range.

[0014] As used herein, the phrase "biological agent" means any compound or molecule that exerts a biological effect. Non-limiting examples of biological agents include proteins (e.g., enzymes), substrates of proteins (e.g., molecules or compounds such as sugars, lipids, fats, polypeptides or proteins, nucleic acids, or others), and other biomolecules.

[0015] As used herein, the phrase "bioprocessing assay" means any assay that receives a biological sample as an input and performs one of a variety of potential operations on the sample, including but not limited to dilution, concentration, separation, mixing, digestion, constitution, reconstitution, aggregation, identification, ontology, sequencing, formulation, etc. Non-limiting examples of bioprocessing assays include peptide mapping, peptide monitoring, proteomics, protein quantification, glycan characterization, glycomics, and glycoprotein characterization.

[0016] As used herein, the phrase "nested conical or frustoconical interface" means the boundary between the top conical or frustoconical surface and the bottom conical or frustoconical surface of the present disclosure. A nested conical or frustoconical interface is formed when the top conical or frustoconical surface and the bottom conical or frustoconical surface are sufficiently close to each other (e.g., no more than 1 μm - 30 μm) to enable the production of the thin film of the present disclosure.

[0017] As used herein, when referring to elements of the devices of the present disclosure, the phrase "operably connected" means that particular elements physically or functionally communicate, attach, or otherwise connect in a manner such that they cooperate to achieve their intended function. Elements that are "operably connected" can be connected directly, indirectly, physically, or remotely.

[0018] As used herein, the term "reaction rate" refers to the rate at which an enzymatic reaction occurs between an enzyme and its substrate molecules (e.g., polypeptides, proteins, nucleic acids, sugars, or lipids). In certain embodiments, the reaction rate can be measured by the rate of accumulation of the concentration of the reaction product or by the rate of depletion of the substrate (i.e., reactant) per unit time. The reaction rate can refer to the rate of a single reaction between a substrate and an enzyme, the combined rate of individual sub-reactions within a reaction chain, or the rate at which a given sub-reaction within a reaction chain occurs. For example, the total reaction rate of a substrate-enzyme reaction can be divided into: (1) the rate of binding between the substrate and the enzyme, designated k on , and the catalytic rate constant of the enzyme itself, designated k cat . At high substrate concentrations, k cat tends to be rate-limiting, while at lower substrate concentrations, the diffusion process significantly reduces k on .

[0019] As used herein, the term "sample injection device" refers to any device configured to deliver (e.g., inject) a sample (such as a liquid sample containing an enzyme and a substrate) to a device of the present disclosure or a component thereof at a specified volume and rate and for a specified duration. Generally, the sample injection device can include a device for containing the sample such as a sample collection structure, a device for measuring and regulating the sample temperature (e.g., a thermometer, a temperature regulator, and a heater element), a device for pumping the sample to the device (e.g., an electric pump and tubing), and optionally a device for removing the sample from the device.

[0020] As used herein, the term "substrate" refers to a biomolecule, complex, or aggregate (e.g., polypeptide, protein, nucleic acid, sugar, or lipid) that acts as a reactant in a chemical reaction involving an enzyme or an enzyme complex. For example, in a chemical reaction involving a protease (e.g., trypsin) and a target molecule to be digested (e.g., any polypeptide or protein), trypsin is the enzyme and the target molecule to be digested is the substrate.

[0021] As used herein, the terms "substrate-enzyme system" and "SES" refer to any mixture containing an enzyme and its natural substrate (e.g., peptide, protein, nucleic acid, sugar, or lipid). Non-limiting examples of SESs include bulk aqueous solutions or thin films containing any amount of an enzyme and its substrate.

[0022] As used herein, when referring to a frustoconical surface, the term "upper base" refers to the base of a frustoconical shape (e.g., frustum) having a smaller diameter. A frustum is essentially a cone or pyramid in which a portion of the cone or solid is cut by a plane parallel to the base of the cone or pyramid, thereby effectively creating a second, smaller base in the cone or pyramid. The "lower base" of a frustoconical shape is the base having the larger diameter (i.e., the base of the cone or pyramid from which the frustoconical shape is created). BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figures 1A to 1D A schematic view showing a top base member of an apparatus for producing the compression and hybrid films of the present disclosure, the top base member having a convex frustoconical surface.( Figure 1A )A top view of a top base member having a cylindrical surface with a central protrusion, the cylindrical surface being operatively connectable to a fixed linear actuator. The protruding cylindrical surface can be assembled to attach to the linear actuator. The lateral protruding surface has holes that can be fitted with one or more heating elements for a heating assembly.( Figure 1B )A lateral cross-section of the top base member showing the frustoconical surface. The diagonal corresponds to the solid interior of the top base member. The central hole can be used as an attachment site for the fixed linear actuator.( Figure 1C )A three-dimensional (3D) view of the top base member having a frustoconical surface.( Figure 1D )A 3D view of a cross-section of the top base member having a frustoconical surface. All measurements are shown in inches.

[0024] Figures 2A to 2B A schematic view showing a top base member of an apparatus for producing the compression and hybrid films of the present disclosure, the top base member having a convex conical surface.( Figure 2A )A top view of a top base member having a cylindrical surface with a central protrusion, the cylindrical surface being operatively connectable to a fixed linear actuator. The protruding cylindrical surface can be assembled to attach to the actuator. Four holes around the protruding cylindrical surface can be assembled to accommodate one or more heating elements for a heating assembly.( Figure 2B )A side view of the conical top base member. All measurements are shown in inches.

[0025] Figures 3A to 3D A schematic view showing a bottom base member of an apparatus for producing the compression and hybrid films of the present disclosure, the top base member having a concave frustoconical surface.( Figure 3A)Top view of a bottom base member having a concave frustoconical surface. The central circle corresponds to the upper base of the frustoconical surface (i.e., the bottom inside the bottom base member). The two lateral holes correspond to the housings that are assembled to accommodate one or more heating elements for the heating assembly.( Figure 3B )Side view of a cross-section of a bottom base member having a concave frustoconical surface. The diagonal corresponds to the solid interior of the bottom base member. The hole at the top of the figure (i.e., the side of the bottom base member) is connected via an internally threaded hole to the upper base of the frustoconical surface of the bottom base member, and this hole corresponds to the fluid inlet port and is used to deliver a biological sample onto the frustoconical surface of the bottom base member via a fluid inlet channel.( Figure 3C )Side view of a bottom base member having a concave frustoconical surface.( Figure 3D )Lateral cross-section of a bottom base member having a concave frustoconical surface, showing the fluid inlet port through which a biological sample is guided onto the frustoconical surface of the bottom base member. All measurements are shown in inches.

[0026] Figure 4 Schematic diagram showing an isometric view of a bottom base member having a concave conical surface.

[0027] Figures 5A to 5C Schematic 3D diagram showing an assembly including a top base member and a bottom base member having a frustoconical surface.( Figure 5A )The top base member shown hovering above the bottom base member.( Figure 5B )The top base member shown nested inside the bottom base member.( Figure 5C )Cross-section of the top base member shown nested inside the bottom base member.

[0028] Figures 6A to 6C Schematic 3D diagram showing an assembly including a top base member and a bottom base member having a conical surface.( Figure 6A )The top base member shown hovering above the bottom base member.( Figure 6B )The top base member shown nested inside the bottom base member.( Figure 6C )Cross-section of the top base member shown nested inside the bottom base member.

[0029] Figures 7A to 7B Illustration and image showing an apparatus for producing the hybrid film of the present disclosure.( Figure 7A)Schematic diagram of a representative device of the present disclosure. In this schematic diagram, a solution containing a mixture of a substrate-enzyme system (SES) is injected through a fluid port in the top base member and stays on the bottom conical surface. A force is applied between the top base member and the bottom base member to bring the top conical surface and the bottom conical surface into close proximity to produce a nested conical interface, in which a thin film containing SES is formed.( Figure 7B )Photograph of a representative device of the present disclosure. The device includes a top base member and a bottom base member having conical surfaces. The top base member is attached to a linear actuator that brings the top base member and the bottom base member into close proximity. The top member also includes a cartridge heater nested inside a heater bore in the top base member, and the heater is operably connected to a relay circuit including a thermistor and a temperature regulator.

[0030] Figures 8A to 8B Is a series of graphs showing the use of pressed films and digesting bovine fibrinogen with trypsin. These pressed films were prepared using static mixing or bulk solutions (i.e., solutions containing SES) prepared from the same volume of starting materials.( Figure 8A )Lineweaver-Burk plot of the "DIQYLPLIK" peptide (SEQ ID NO:1) of bovine fibrinogen, which was digested using preparations containing 1 mg / mL or 8 mg / mL pressed films or control bulk solutions containing the trypsin-fibrinogen system.( Figure 8B )Lineweaver-Burke plot of the "AIQISYNPDQPSKPNNIESATK" peptide (SEQ ID NO:2) of bovine fibrinogen, which was digested using preparations containing 1 mg / mL or 8 mg / mL pressed films or control bulk solutions containing the trypsin-fibrinogen system. The x-intercept of the Lineweaver-Burke plot is equal to -1 / K m , where K m is the Michaelis constant; the y-intercept corresponds to 1 / V max , where V max is the maximum rate of the enzyme-catalyzed reaction; and the slope of the graph is proportional to K m / V max These graphs demonstrate that V0 is independent of the substrate concentration in the film and show that for certain peptides, the use of film preparations significantly increases the V of the reaction compared to control bulk solutions max . Compared to 2.2 counts / s in the bulk solution, the V maxwas 11.1 counts / s. Compared to 0.3 counts / s in the bulk solution, for AIQISYNPDQPSKPNNIESATK (SEQ ID NO:2) in the film, V max was 12.6 counts / s.

[0031] Figure 9 is a bar graph showing the peptide abundances of multiple digested peptide fragments of antibodies digested with trypsin using static or dynamic films or a control bulk solution over a 16-minute period. The peptide fragments tested include, from left to right: (-)DVLMTQTPLSLPVSLGDQASISCR(S) (SEQ ID NO:3), (R)ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK(D) (SEQ ID NO:4), (R)QNGVLNSWTDQDSK(D) (SEQ ID NO:5), (R)VEAEDLGVYYCFQGSHVOKTFGAGGTK(L) (SEQ ID NO:6), (K)TSTSPIVK(S) (SEQ ID NO:7), (K)APQVYTIPPPKEQMAK(D) (SEQ ID NO:8), (R)VNSAAFPAPIEK(T) (SEQ ID NO:9), (K)DVLTITLTPK(V) (SEQ ID NO:10), (K)SQVFLK(M) (SEQ ID NO:11), and (K)QYFAYWGQGTLVTVSAAK(T) (SEQ ID NO:12). The amino acid positions indicated in parentheses are not part of the peptide fragment but correspond to the preceding and following amino acids from the full-length polypeptide from which they were generated ("(-)" indicates no preceding or following amino acid at the indicated position).

[0032] Figures 10A to 10D is a plot showing the digestion kinetics of the selected peptides described above using static film preparations, dynamic film preparations, and a control bulk solution over a 16-minute period, as measured by the abundance of each peptide species, which includes (R)QNGVLNSWTDQDSK(D) (SEQ ID NO:5) ( Figure 9 )、(K)APQVYTIPPPKEQMAK(D) (SEQ ID NO:8) ( Figure 10A )、(R)VNSAAFPAPIEK(T) (SEQ ID NO:9) ( Figure 10B )、and (R)VEAEDLGVYYCFQGSHVOKTFGAGGTK(L) (SEQ ID NO:6) ( Figure 10C )、and (R)VEAEDLGVYYCFQGSHVOKTFGAGGTK(L) (SEQ ID NO:6) ( Figure 10D)。These curves demonstrate that the digestion of the antibody was improved using static and dynamic membranes compared to the control solution, with the dynamic membrane showing the fastest digestion kinetics.

[0033] Figure 11 Deconvoluted mass spectra are shown, which show large peptide fragments from trypsin digestion in bulk solution (upper panel), large peptide fragments from digestion in thin films (middle panel), and large peptide fragments from digestion in dynamic thin films (lower panel). In thin film digestion and dynamic thin film digestion, the abundance of the large 36 kDa fragment was significantly reduced. Instead, these digests were characterized by many smaller fragments in the 5 kDa - 10 kDa range. This indicates that under thin film conditions, the intact protein was better digested into smaller intermediates.

[0034] Figure 12 is a bar graph showing the peptide abundances of the histograms, which show the number of peptides with a given abundance in each digestion technique. Thin films and dynamic thin films generally produced more peptides, but especially relatively low abundance peptides with intensities below 10,000.

[0035] Figures 13A to 13D Curves are shown that illustrate the yield of four peptides QVQLK (SEQ ID NO:13; Figure 13A )、DVLTITLTPK(SEQID NO:14; Figure 13B )、VTCVVVDISKDDPEVQFSWFVDDVEVHTAHTQPR(SEQ ID NO:15; Figure 13C ) and EEQFNSTFR(SEQ ID NO:16; Figure 13D ) from the one-pot thin film digestion of a reference monoclonal antibody (Waters mass number calibration standard) as a function of membrane thickness. DETAILED DESCRIPTION

[0036] In certain embodiments, methods for accelerating the reaction rate of a substrate-enzyme system (SES) are disclosed herein, including preparing a pressed and mixed thin film comprising a predetermined ratio of a substrate (e.g., polypeptide, protein, nucleic acid, sugar or lipid) and an enzyme (e.g., protease, nuclease, glycosidase, lipase, kinase, phosphatase, etc.) that acts on the substrate. Such methods can be used, for example, in sample preparation for a variety of bioanalytical or bioprocessing assays (e.g., peptide mapping, peptide monitoring, proteomics, protein quantification, glycan characterization, glycomics, and glycoprotein characterization). Devices for producing the pressed and mixed thin films are also disclosed. The following sections describe the invention in more detail.

[0037] Reaction Rate in Substrate-Enzyme System

[0038] A substrate-enzyme system (SES) is a simple or complex mixture of an enzyme and its corresponding substrate (e.g., polypeptide, protein, nucleic acid, sugar, or lipid), such as a solution containing the substrate and the enzyme. The reaction rate in the SES is given by two independent rate constants, namely the binding rate of the substrate and enzyme complex, called k on , and the catalytic rate constant of the enzyme, called k cat . At high substrate concentrations, k cat tends to be rate-limiting, while at lower substrate concentrations, the diffusion process significantly reduces k on .

[0039] The most common industrial enzymatic reactions are carried out in a bulk reaction system, usually in batch reactions, where the overall reaction rate is largely limited by the diffusion of the enzyme and the substrate (i.e., k on ). To increase the reaction rate of such reactions, the use of microdroplets has been attempted. The acceleration of the reaction rate in microdroplets can be largely attributed to the spatial confinement and the increase in the k on rate caused by droplet dehydration. The limiting characteristic of microdroplets is their inherent short lifespan. On the other hand, thin films exhibit a lifespan many orders of magnitude longer than that of microdroplets, thus providing a potential avenue for accelerating the reaction rate in the SES. In certain embodiments, thin films and methods for their preparation are disclosed herein for accelerating the reaction rate of the SES, thereby improving the efficiency and productivity of sample preparation in bioprocessing and bioanalytical assays.

[0040] Thin Film

[0041] A thin film is a material substrate formulated as a thin layer, typically having a thickness in the range between less than one nanometer (nm) to several micrometers (μm). These films have a wide range of applications, including but not limited to those for magnetic recording media, semiconductor devices, integrated passive devices, light-emitting diodes (LEDs), optical coatings, cutting tool coatings, solar cells, storage batteries, and drug delivery systems. Methods for producing thin films include but are not limited to spray deposition, spin casting, and physical pressing. Spin casting devices are common and provide a simple implementation, as a certain volume of liquid can be placed on a rotating flat platform and rotated to produce a thin film. However, pressing provides an alternative that allows for improved control of the film thickness and offers the possibility of mixing by using repeated pressing. This technique is similar to the method employed in a droplet array sandwich. This effect is driven by the high cohesion of the aqueous solvent, and this method can be easily implemented into a lab-on-a-chip workflow.

[0042] Sample Preparation

[0043] Accordingly, in certain embodiments, a method of preparing a biological sample (e.g., a protein sample) in a thin film for use in a biological processing assay (e.g., an assay for the isolation, purification, identification, and / or characterization of a biological sample such as a protein) is disclosed herein. In certain embodiments, the method includes preparing the biological sample by combining a target enzyme with a substrate (e.g., a polypeptide, protein, nucleic acid, sugar, or lipid) at a predetermined ratio to produce a substrate-enzyme mixture (e.g., SES). Non-limiting examples of enzymes suitable for use in conjunction with the disclosed method include proteases, glycosylating enzymes, and phosphatases, among others. In certain embodiments, the protease is trypsin, chymotrypsin, immunoglobulin-degrading enzyme (IgDE), immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS), Asp-N protease, or Lys-C protease. In certain embodiments, the glycosylating enzyme is peptide:N-glycosidase F (PNGase F). In certain embodiments, the phosphatase is alkaline phosphatase (ALP). In certain embodiments, the sample is prepared by mixing the enzyme and the substrate in an aqueous solvent (e.g., phosphate-buffered saline (PBS), TRIS buffer, ammonium bicarbonate, sodium chloride, sodium sulfate, etc.) using known methods. In certain embodiments, the predetermined ratio of enzyme to substrate is about 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, or 1:200 (moles of enzyme:moles of substrate). The substrate-enzyme mixture can be included in the aqueous solvent at various concentrations, including but not limited to between 0.1 mg / mL and 20 mg / mL (e.g., 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, and 20 mg / mL). In certain embodiments, the total volume of the aqueous solvent containing the substrate-enzyme mixture is between 10 μL and 100 μL (10 μL to 100 μL, 15 μL to 95 μL, 20 μL to 90 μL, 25 μL to 85 μL, 30 μL to 80 μL, 35 μL to 75 μL, 40 μL to 70 μL, 45 μL to 65 μL, and 50 μL to 60 μL).In certain embodiments, the substrate-enzyme mixture is prepared by, for example, mixing 12.5 μL of a solution containing a substrate at a concentration of 2 mg / mL with 12.5 μL of a solution containing an enzyme at a concentration of 0.1 mg / mL. In certain embodiments, the substrate-enzyme mixture further comprises CaCl2 (e.g., 4 mM), a surfactant (e.g., RAPIGEST). TM , e.g., 0.1%-0.5%), and guanidine hydrochloride.

[0044] In certain embodiments, the viscosity of a sample containing the substrate-enzyme mixture and a solvent is adjusted using known methods to achieve a desired consistency. For example, in certain embodiments, the solution is adjusted to have a viscosity of 0.1 mPa·s to 2.0 mPa·s (e.g., 0.1 mPa·s to 2.0 mPa·s, 0.2 mPa·s to 1.9 mPa·s, 0.3 mPa·s to 1.8 mPa·s, 0.4 mPa·s to 1.7 mPa·s, 0.5 mPa·s to 1.6 mPa·s, 0.6 mPa·s to 1.5 mPa·s, 0.7 mPa·s to 1.4 mPa·s, 0.8 mPa·s to 1.3 mPa·s, 0.9 mPa·s to 1.2 mPa·s, and 1.0 mPa·s to 1.1 mPa·s). Various other parameters of the sample can be optimized using conventional methods to achieve favorable properties, such as the pH, temperature, homogeneity, melting point, boiling point, and saturation of the sample.

[0045] Thin Film Production

[0046] In certain embodiments, once a sample containing a substrate-enzyme mixture is prepared, it is deposited in a predetermined amount (e.g., between 10 μL and 100 μL) on a conical or frustoconical surface, such as the conical or frustoconical surface of the device disclosed herein. The sample containing the mixture is deposited on the surface using a variety of conventional methods and devices, e.g., using a sample injection device operatively connected to a device having two nested conical or frustoconical surfaces. The mixture is pressed between the two surfaces by applying a predetermined force between the bottom conical or frustoconical surface on which the sample is deposited and the top conical or frustoconical surface, thereby forming a thin film containing the substrate-enzyme mixture. In certain embodiments, the predetermined force is between 3 pounds and 23 pounds (e.g., 3 pounds to 20 pounds, 4 pounds to 19 pounds, 5 pounds to 18 pounds, 6 pounds to 17 pounds, 7 pounds to 16 pounds, 8 pounds to 15 pounds, 9 pounds to 14 pounds, 10 pounds to 13 pounds, and 11 pounds to 12 pounds). In certain embodiments, the predetermined force is between 10 pounds and 15 pounds (e.g., 10 pounds to 15 pounds, 11 pounds to 14 pounds, and 12 pounds to 13 pounds). The pressing of the top and bottom surfaces creates a nested conical or frustoconical interface in which the mixture is molded, and this interface partly determines the thickness of the resulting thin film. In certain embodiments, the pressing is performed once and for a predetermined duration (e.g., static pressing). In certain embodiments, the force is applied and maintained for a duration between 3 s and 5 s (e.g., 3.0 s to 5.0 s, 3.1 s to 4.9 s, 3.2 s to 4.8 s, 3.3 s to 4.7 s, 3.4 s to 4.6 s, 3.5 s to 4.5 s, 3.6 s to 4.4 s, 3.7 s to 4.3 s, 3.8 s to 4.2 s, and 3.9 s to 4.1 s). Repeated pressing of the top and bottom surfaces for a predetermined duration (e.g., active dynamic pressing) can also be performed to facilitate mixing of the enzyme and substrate. Such dynamic pressing can include applying and withdrawing the force at predetermined intervals and for predetermined durations. For example, in certain embodiments, the force is applied for a duration of 3 seconds to 5 seconds, followed by withdrawing the force for a duration between 1 s and 2 s (e.g., 1.0 s to 2.0 s, 1.1 s to 1.9 s, 1.2 s to 1.8 s, 1.3 s to 1.7 s, and 1.4 s to 1.6 s). In certain embodiments, in an active dynamic pressing scheme, the application and withdrawal of the force are performed over a total duration of about 30 s. In certain embodiments, the pressing step (e.g., static or dynamic pressing) is performed for a time sufficient to produce a thin film having a desired thickness, density, consistency, compactness, viscosity, or SES reaction rate acceleration. In certain embodiments, the thickness of the thin film is not greater than about 100 μm, about 90 μm, about 80 μm, about 70 μm, about 60 μm, about 50 μm, about 40 μm, about 30 μm, about 20 μm, or about 10 μm. In certain embodiments, the thin film has a thickness not greater than about 50 μm.

[0047] In certain embodiments, the thin film accelerates the reaction rate of the SES by increasing the binding rate (K on ) of the enzyme to the substrate. In certain embodiments, the thin film accelerates the reaction rate of the SES by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 2,000-fold, 3,000-fold, 4,000-fold, 5,000-fold, 6,000-fold, 7,000-fold, 8,000-fold, 9,000-fold, 10,000-fold or more compared to the reaction rate of the SES in the control bulk solution. In certain embodiments, the control bulk solution has the same volume as the starting volume of the aqueous sample used to prepare the thin film. In certain embodiments, the control bulk solution has the same temperature as the starting temperature of the aqueous sample used to prepare the thin film and / or the temperature of the thin film. In certain embodiments, the reaction of the SES in the thin film proceeds for the same duration as the reaction of the SES in the bulk control solution. In certain embodiments, the reaction of the SES (e.g., in the thin film or in the control bulk solution) is terminated using chemical means (e.g., guanidine hydrochloride).

[0048] Equipment for Producing Thin Film

[0049] In certain embodiments, disclosed herein is an apparatus configured to produce a thin film containing a substrate-enzyme system (SES). In certain embodiments, such an apparatus includes means for receiving an aqueous sample (e.g., a fluid inlet port), means for containing the sample (e.g., a bottom conical or frustoconical surface), means for controlling the temperature of the sample on the surface (e.g., a thermometer, a temperature regulator, and a heating element), and means for pressing the sample (e.g., a top conical or frustoconical surface operably connected to a linear actuator configured to generate a predetermined force). In certain embodiments, the means for receiving the aqueous sample, the means for containing the sample, the means for controlling the sample temperature, and the means for pressing the sample are operably connected to each other. In certain embodiments, the means for receiving the aqueous sample is physically connected to the means for containing the sample. In certain embodiments, the means for containing the sample is physically connected to the means for controlling the sample temperature. In certain embodiments, the means for pressing the sample is physically connected to the linear actuator.

[0050] Top Base Member and Bottom Base Member

[0051] In certain embodiments, an apparatus configured to produce a film comprising SES according to the present disclosure includes a top base member (see, for example, Figures 1A to 1D , Figures 2A to 2B , Figures 5A to 5C , Figures 6A to 6C and Figure 7B ) and a bottom base member (see, for example, Figures 3A to 3D , Figure 4 , Figures 5A to 5C , Figures 6A to 6C and Figure 7B ), the top base member and the bottom base member having a conical surface (see, for example, Figures 2A to 2B and Figure 4 ) or a frustoconical surface (see, for example, Figures 1A to 1D and Figures 3A to 3D ). The top base member includes on one side a vertically oriented, downward-facing, and convex conical ( Figure 2B and Figure 7B ) or frustoconical surface ( Figures 1B to 1D ), the conical or frustoconical surface being attached to the top base member at the base of the conical surface or the bottom base of the frustoconical surface (see, for example, Figures 1A to 1D and Figures 2A to 2B ). On the other hand, in certain embodiments, the upward-facing side of the top base member is a raised cylindrical surface on the top base member (see, for example, Figures 1A to 1D , Figures 2A to 2B , Figures 5A to 5C , Figures 6A to 6C and Figure 7B ), the cylindrical surface being configured to be operatively connected to a fixed linear actuator configured to move along a vertical axis ( Figures 7A to 7B ). In certain embodiments, the bottom base member is a hollow, vertically oriented, and concave conical ( Figure 4 , Figure 6A and Figure 6C) or a cylinder with a frustoconical surface. In certain embodiments, the bottom base member is operably connected to a fixed linear actuator configured to move along a vertical axis and bring the top and bottom conical or frustoconical surfaces into contact or close enough proximity to produce a film. In certain embodiments, both the top and bottom base members are operably connected to a fixed linear actuator. In certain embodiments, only the top base member is operably connected to a fixed linear actuator. In certain embodiments, only the bottom base member is operably connected to a fixed linear actuator. Although the size of the device can be scaled arbitrarily, in certain embodiments, the top and / or bottom conical or frustoconical surfaces of the device can each have a slant height of 3 cm to 15 cm (e.g., 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, and 15 cm). In certain embodiments, the top conical or frustoconical surface and the bottom conical or frustoconical surface each have an apex angle of 45°. For the case of using a frustoconical surface instead of a conical surface, the top frustoconical surface and the bottom frustoconical surface include an upper base with a diameter of 1 mm to 10 mm (e.g., 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, and 10 cm). Regarding the fluid sample used to produce the film of the present disclosure, the bottom conical or frustoconical surface is configured to accommodate a fluid volume between at least 10 μL and 100 μL (e.g., 10 μL to 100 μL, 15 μL to 95 μL, 20 μL to 90 μL, 25 μL to 85 μL, 30 μL to 80 μL, 35 μL to 75 μL, 40 μL to 70 μL, 45 μL to 65 μL, and 50 μL to 60 μL).

[0052] Sample Delivery

[0053] As described above, in certain embodiments, the devices of the present disclosure include means for receiving an aqueous sample, such as a fluid inlet port operatively connected to a top or bottom conical or frustoconical surface. In certain embodiments, the fluid inlet port is operatively connected to the bottom conical or frustoconical surface. In certain embodiments, the fluid inlet port is operatively connected to the top conical or frustoconical surface. The fluid inlet port can have any configuration as long as it is capable of supplying a fluid sample (e.g., a fluid sample containing SES) to the top or bottom base member at a predetermined volume, predetermined time, and / or at a predetermined temperature. In certain embodiments, the fluid inlet port is operatively connected (e.g., via a conduit) to a fluid source containing SES in an aqueous solvent and optionally operatively connected to a fluid pump configured to move the fluid sample from the fluid source to the fluid inlet port. In certain embodiments, the fluid inlet port guides the fluid sample from the fluid source to and deposits it on the bottom conical or frustoconical surface via a fluid inlet channel. In certain embodiments, the fluid inlet channel is operatively connected to the top or bottom conical or frustoconical surface. Regardless of whether the fluid inlet channel is connected to the top surface or the bottom surface, the force of gravity aids in depositing the fluid sample on the bottom conical or frustoconical surface, which serves as means for containing the fluid sample prior to pressing and optional mixing of the fluid sample.

[0054] Pressing and Mixing

[0055] In certain embodiments, the devices of the present disclosure include means for pressing a fluid sample to produce a pressed and optionally mixed film from the sample. In certain embodiments, the means for pressing the fluid sample is a fixed linear actuator operatively connected to the top or bottom base member or both. The linear actuator moves the top or bottom base member or both along a vertical axis such that the top conical or frustoconical surface of the top base member and the bottom conical or frustoconical surface of the bottom base member contact or are in close proximity (e.g., within 1 μm - 30 μm of each other) to produce a nested conical or frustoconical interface, wherein the fluid sample is physically pressed to produce a film containing SES. In certain embodiments, the linear actuator is configured to produce a predetermined force between the top conical or frustoconical surface and the bottom conical or frustoconical surface, the predetermined force being between 3 pounds and 20 pounds (e.g., 3 pounds to 20 pounds, 4 pounds to 19 pounds, 5 pounds to 18 pounds, 6 pounds to 17 pounds, 7 pounds to 16 pounds, 8 pounds to 15 pounds, 9 pounds to 14 pounds, 10 pounds to 13 pounds, and 11 pounds to 12 pounds). Additionally, the linear actuator can be vertically positioned at a slight offset angle (e.g., 10° - 30°) such that the actuator produces a slight lateral (i.e., horizontal) movement to help reduce the surface tension of the film.

[0056] Temperature Control

[0057] As described above, in certain embodiments, the disclosed device includes means for controlling the temperature of a sample (e.g., a fluid sample or a thin film) on one or more surfaces of the device. Thus, temperature control of the device can be exemplified by using one or more (e.g., 1, 2, 3, 4, or more) heating elements (e.g., cartridge heaters or infrared lasers) that are operatively connected to a top or bottom base member. In certain embodiments, the one or more heating elements are operatively connected to one or more thermistors, relay circuits, and temperature regulators. The temperature range suitable for preparing the thin films disclosed herein can vary depending on the specific SES preparation but can include temperatures between 20°C and 80°C.

[0058] Surface Properties

[0059] According to the present disclosure, the surfaces of the devices disclosed herein (e.g., top and bottom conical or frustoconical surfaces) can be any material known to be suitable for producing thin films. For example, the surfaces can be made of stainless steel, poly(methyl acrylate), or glass, among others.

[0060] Additionally, the top and / or bottom conical or frustoconical surfaces of the devices disclosed herein can be modified by physical or chemical means in order to obtain optimal surface properties for preparing thin films as disclosed herein. For example, the top and / or bottom conical or frustoconical surfaces can be physically modified by deposition-based coating with, for example, polyethylene glycol (PEG), phenyl, or C2. Additionally or alternatively, the top and / or bottom conical or frustoconical surfaces can be functionalized by chemical means, such as by silanization and then functionalized with PEG, phenyl, C2, or other modifiers.

[0061] Bioprocess Assay

[0062] In certain embodiments, the methods and devices disclosed herein are for the preparation of biological samples in a variety of bioprocessing assays. As described above, the time required for the preparation reaction of analytical techniques in a bioprocessing space can be delayed due to reaction rate limitations, thus hindering an efficient workflow. These delays impede the acquisition of real-time results required for feedback loop regulation of bioreactor design. Thus, the disclosed methods and devices provide an improvement in the sample preparation for bioprocessing / analytical assays by reducing the rate of the preparation reaction. Non-limitingly, the disclosed methods and devices are suitable for use in combination with a variety of bioprocessing assays, including peptide mapping, peptide monitoring, proteomics, protein purification, glycan characterization, glycomics, and glycoprotein characterization.

[0063] Analysis of the primary structure of proteins is necessary for protein characterization. Peptide mapping (also known as peptide mass fingerprinting) is a valuable method that combines positional quantitative information with topological and domain information of proteins. In particular, peptide mapping is a useful procedure and a key goal in many genome sequencing projects as well as biomedical and biopharmaceutical research efforts. In a typical peptide mapping workflow, the isolated protein is fragmented by enzymatic digestion (e.g., with proteases such as trypsin, chymotrypsin, pepsin, glutamyl endopeptidase, etc.), and then separated and analyzed using methods such as mass spectrometry (MS) or high-performance liquid chromatography (HPLC). The masses of the digested peptide fragments are then compared to a database containing reference protein sequences and their corresponding fragments. The comparison of the masses of unknown protein fragments with those of known protein fragments aids in the identification and characterization of the unknown protein. Thus, the disclosed methods and devices are suitable for use in conjunction with peptide mapping techniques.

[0064] Relatedly, peptide monitoring is commonly used to analyze the quality of biotherapeutic drugs (e.g., peptide or protein therapeutics). Proteolytic digests are often analyzed as a monitoring technique for the protein composition of biotherapeutic drugs. The high costs associated with meeting regulatory guidelines for biotherapeutic drug safety have renewed interest in effective methods for reducing costs and increasing productivity during the manufacturing process.

[0065] In addition, proteomics analysis often involves analyzing proteins using electrophoresis separation, MS, and / or liquid chromatography. In various applications of these methods, proteins in complex mixtures are fragmented and labeled, and then the labeled peptide fragments are chromatographically separated and analyzed by MS. The efficiency and throughput of such analysis depend at least in part on the efficiency of protein preparation (e.g., enzymatic digestion of protein samples). Thus, the disclosed methods and devices can be used in conjunction with proteomics analysis to improve its efficiency and cost.

[0066] Protein purification is another approach to proteins, aimed at isolating the target protein from complex biological samples (e.g., protein mixtures, cells, tissues, etc.) for, e.g., sample preparation and / or characterization of the protein's structure, function, quantity, etc. Typical protein purification and quantification workflows include isolating the target protein, denaturing the protein's tertiary and secondary structures for efficient digestion, and enzymatically cleaving the protein at predicted amino acid residues to reduce sample complexity, thereby improving the sensitivity and specificity of proteomics analysis.

[0067] Characterizing and monitoring the glycan populations of biotherapeutic proteins (e.g., glycoprotein profiling, released glycan characterization and monitoring, subunit analysis, monosaccharide / sialic acid composition analysis, and glycopeptide mapping) has presented technical and logistical challenges to the biopharmaceutical industry, at least in part due to the limited efficiency and productivity of the preparative reactions of glycoprotein-containing preparations. As in proteomic analysis, the analysis of glycan and glycoprotein samples typically involves the preparative digestion of glycoproteins for subsequent analysis and characterization, which can become a bottleneck when the preparative reaction is rate-limiting. Thus, the disclosed methods and devices can be advantageously used in conjunction with the characterization and monitoring of glycans and glycoproteins to improve the efficiency of such analyses.

[0068] Examples

[0069] The following examples are presented to provide a description to those of ordinary skill in the art of how the compositions and methods described herein can be used, prepared, and evaluated, and are intended to illustrate the disclosure purely by way of example and not to limit the scope that the inventors regard as their disclosure.

[0070] Example 1: Trypsin Digestion of Bovine Fibrinogen Using a Pressed Film

[0071] To evaluate the enzymatic digestion efficacy of a substrate-enzyme system (SES) prepared on the compression films of the present disclosure, the following steps were carried out. Bulk-phase or isothermal film digestion was used to digest bovine fibrinogen. Trypsin was prepared at 0.1 mg / mL in 100 mM ammonium bicarbonate and 4 mM calcium chloride. Bovine fibrinogen was prepared at 1 mg / mL, 2 mg / mL, 4 mg / mL, and 8 mg / mL in 100 mM ammonium bicarbonate containing 0.2% RAPIGEST TM surfactant. The reactions were initiated by mixing 12.5 μL of each solution and quenched by adding 200 μL of guanidine hydrochloride. Peptide abundances were determined by liquid chromatography-mass spectrometry (LC-MS) of the resulting mixtures. Films were produced using the device disclosed herein. The reciprocal of the initial reaction rate V0 of the peptides DIQYLPLIK (SEQ ID NO:1) and AIQISYNPDQPSKPNNIESATK (SEQ ID NO:2) was plotted against the reciprocal of the fibrinogen concentration to obtain a Lineweaver-Burke plot ( Figure 8A and Figure 8B ). The x-intercept of the Lineweaver-Burke plot is equal to -1 / K m , where K m is the Michaelis constant; the y-intercept corresponds to 1 / V max , where V max is the maximum rate of the enzyme-catalyzed reaction; and the slope of the curve is related to K m / V maxProportional. These curves demonstrate that V0 is independent of the substrate concentration in the thin film and show that for certain peptides, the use of thin film preparations significantly increases the V of the reaction compared to the control bulk solution. max . The V calculated for DIQYLPLIK (SEQ ID NO:1) in the thin film was 11.1 counts / s compared to 2.2 counts / s in the bulk solution. max . The V for AIQISYNPDQPSKPNNIESATK (SEQ ID NO:2) in the thin film was 12.6 counts / s compared to 0.3 counts / s in the bulk solution. max

[0072] Example 2: Trypsin Digestion of Monoclonal Antibody Using a Pressed Film

[0073] The Waters intact monoclonal antibody assay standard was prepared at 2 mg / mL in 100 mM ammonium bicarbonate containing 0.2% RAPIGEST TM surfactant. Trypsin was prepared at 0.1 mg / mL in 100 mM ammonium bicarbonate and 4 mM calcium chloride. Enzymatic digestion was initiated by mixing 12.5 μL of each solution and quenched with 200 μL of 6 M guanidine hydrochloride. Peptide abundances were determined by LC-MS of the resulting mixture. Peptide abundances of multiple digested peptide fragments of the digested antibody were measured using static or dynamic thin films or the control bulk solution over a 16-minute period. The peptide fragments tested included: (-)DVLMTQTPLSLPVSLGDQASISCR(S)(SEQID NO:3), (R)ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK(D)(SEQ ID NO:4), (R)QNGVLNSWTDQDSK(D)(SEQ ID NO:5), (R)VEAEDLGVYYCFQGSHVOKTFGAGGTK(L)(SEQ ID NO:6), (K)TSTSPIVK(S)(SEQ ID NO:7), (K)APQVYTIPPPKEQMAK(D)(SEQ ID NO:8), (R)VNSAAFPAPIEK(T)(SEQ ID NO:9), (K)DVLTITLTPK(V)(SEQ ID NO:10), (K)SQVFLK(M)(SEQID NO:11), and (K)QYFAYWGQGTLVTVSAAK(T)(SEQ ID NO:12)( Figure 9)。The amino acid positions indicated in parentheses are not part of the peptide fragment but correspond to the preceding and following amino acids from the full-length polypeptide from which they are derived ("(-)" indicates no preceding or following amino acid at the indicated position). In all cases, the thin film reaction system improved protein digestion efficacy compared to the bulk solution reaction system. For certain peptide fragments, the relative peptide abundance was significantly higher when using dynamic mixing compared to static mixing ( Figure 9 and Figures 10A to 10D ).

[0074] Deconvolution mass spectrometry analysis of the digested antibody showed that the abundance of the large 36 kDa fragment was significantly reduced in both static and dynamic thin film digestions compared to bulk solution digestion. Instead, these digests were characterized by many smaller fragments in the 5 kDa - 10 kDa range. This indicates that intact proteins are better digested into smaller intermediates in the thin film system ( Figure 11 ), especially those below 10,000 (peptide abundance) ( Figure 12 ).

[0075] Example 3: Effect of Pressed Film Thickness on Enzymatic Digestion of Monoclonal Antibody

[0076] To determine the effect of the suppression film thickness on the enzymatic digestion of a reference monoclonal antibody (Waters mass number check standard), annular gaskets with inner and outer diameters equal to the edge of the bottom frustoconical plate were produced with thicknesses of 12.5 μm, 25 μm, and 37.5 μm such that the stacking of the gaskets would result in an offset gap being applied on both plates. The reaction was carried out in a one-pot digestion manner, where 5 mM TCEP and the enzyme were added to the antibody in 10 mM ammonium bicarbonate containing 0.2% RAPIGEST TM detergent. The reaction was carried out at 55 °C for 20 minutes, and the resulting peptide abundances of the digested peptides were analyzed by LC-MS. The variation of the yields of four peptides QVQLK (SEQ ID NO:13), DVLTITLTPK (SEQ ID NO:14), VTCVVVDISKDDPEVQFSWFVDDVEVHTAHTQPR (SEQ ID NO:15), and EEQFNSTFR (SEQ ID NO:16) with film thickness is provided in Figures 13A to 13D , demonstrating the effect of the reduced film thickness on the yield. The effect of the thin film with a thickness less than 50 μm was obvious and produced a linear response with the reaction yield.

[0077] Other Embodiments

[0078] Various modifications and variations of the described disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the disclosure. Other embodiments are protected by the claims.

Claims

1. A method for preparing a protein sample in a thin film for a biological processing assay, the method comprising: (a) Combining an enzyme and a protein substrate at a predetermined ratio to produce a substrate-enzyme mixture; (b) Depositing the mixture of step (a) in a bottom conical or frustoconical surface; And (c) Pressing the mixture between the bottom conical or frustoconical surface and a top conical or frustoconical surface by applying a predetermined force between the top conical or frustoconical surface and the bottom conical or frustoconical surface, wherein the top conical or frustoconical surface and the bottom conical or frustoconical surface are vertically oriented and configured to produce a nested conical or frustoconical interface; Thereby forming a thin film containing the protein-enzyme mixture in the nested conical interface.

2. The method according to claim 1, wherein the predetermined ratio of the enzyme and the protein substrate is about 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190 or 1:200 (moles of enzyme: moles of substrate).

3. The method according to claim 1 or 2, wherein the substrate-enzyme mixture is present in solution at a concentration of 0.1 mg / mL to 20 mg / mL.

4. The method according to claim 3, wherein the enzyme mixture is in 10 μL to 100 μL of the solution.

5. The method according to any one of claims 1 to 4, wherein the solution has a viscosity of 0.1 mPa·s to 2.0 mPa·s.

6. The method according to any one of claims 1 to 5, wherein the predetermined force is between 3 pounds and 20 pounds.

7. The method according to claim 6, wherein the predetermined force is between 10 pounds and 15 pounds.

8. The method according to any one of claims 1 to 7, wherein the force is applied and maintained for a duration between 3 s and 5 s.

9. The method according to claim 8, wherein after the application of the force, the force is withdrawn for a duration between 1 s and 2 s.

10. The method according to claim 8 or 9, wherein the application and withdrawal of the force are carried out within a total duration of 30 s.

11. The method according to any one of claims 1 to 10, wherein the method increases the reaction rate between the enzyme and the substrate as compared to the reaction rate between the enzyme and the substrate in the bulk solution.

12. The method according to claim 11, wherein the increase in the reaction rate is due to an increase in the binding rate (K on ) of the enzyme to the substrate.

13. The method according to any one of claims 1 to 12, wherein the enzyme is selected from proteases, glycosylating enzymes, and phosphatases.

14. The method according to claim 13, wherein the protease is trypsin, chymotrypsin, immunoglobulin-degrading enzyme (IgDE), immunoglobulin G-degrading enzyme (IdeS) of Streptococcus pyogenes, Asp-N protease, or Lys-C protease.

15. The method according to claim 13, wherein the glycosidase is peptide:N-glycosidase F (PNGase F).

16. The method according to claim 13, wherein the phosphatase is alkaline phosphatase (ALP).

17. The method according to any one of claims 1 to 16, wherein the bioprocessing assay is liquid chromatography (LC), mass spectrometry (MS), or a combination thereof.

18. The method according to any one of claims 1 to 17, wherein the bioprocessing assay is peptide mapping, peptide monitoring, proteomics, protein quantification, glycan characterization, glycomics, and glycoprotein characterization.

19. An apparatus for producing a thin film, the apparatus comprising: (a) a vertically oriented convex top conical or frustoconical surface, wherein a base of the top conical or frustoconical surface is attached to a top base member; (b) a vertically oriented concave bottom conical or frustoconical surface configured to receive a sample volume, wherein the bottom conical or frustoconical surface is inside a cylindrical bottom base member; (c) a fixed linear actuator configured to move along a vertical axis and operably connected to a raised cylindrical surface on the top base member or operably connected to the bottom base member; (d) a fluid inlet port operably connected to the top conical or frustoconical surface and / or the bottom conical or frustoconical surface; (e) one or more heating elements operably connected to a temperature controller and a relay circuit; (f) a thermistor configured to be placed within a thermistor hole in the raised cylindrical surface of the top base member, the thermistor being operably connected to the temperature controller; wherein the top conical or frustoconical surface and the bottom conical or frustoconical surface are configured to produce a nested conical or frustoconical interface.

20. The apparatus according to claim 19, wherein the thin film is configured for depositing one or more biologic agents on a surface of the thin film.

21. The apparatus according to claim 20, wherein the one or more biologic agents are target proteins.

22. The apparatus according to claim 21, wherein the target protein is an enzyme.

23. The apparatus according to claim 22, wherein the one or more biologic agents further comprise a substrate of the enzyme.

24. The apparatus according to any one of claims 19 to 23, wherein the linear actuator is configured to produce a predetermined force between the top conical or frustoconical surface and the bottom conical or frustoconical surface, the predetermined force being between 3 pounds and 20 pounds.

25. The apparatus according to any one of claims 19 to 24, wherein the linear actuator is configured to produce an angular motion with an offset angle of 10° - 30°.

26. The apparatus according to any one of claims 19 to 25, wherein each of the top and / or bottom conical or frustoconical surfaces has a slant height of 3 cm to 15 cm.

27. The device according to any one of claims 19 to 26, wherein the bottom conical or frustoconical surface has a vertical height of from 2.85 cm to 10 cm.

28. The device according to any one of claims 19 to 27, wherein the top conical or frustoconical surface and the bottom conical or frustoconical surface each have an apex angle of 45°.

29. The device according to any one of claims 19 to 28, wherein the top frustoconical surface and the bottom frustoconical surface include an upper base having a diameter of from 1 mm to 10 mm.

30. The device according to any one of claims 19 to 29, wherein each of the top conical or frustoconical surface and the bottom conical or frustoconical surface is independently composed of a material selected from stainless steel, poly(methyl acrylate), and glass.

31. The device according to any one of claims 19 to 30, wherein the fluid inlet port is operatively connected to a sample injection device.

32. The device according to any one of claims 19 to 31, wherein the top conical or frustoconical surface is actuated while the bottom conical or frustoconical surface is stationary.

33. The device according to any one of claims 19 to 31, wherein the bottom conical or frustoconical surface is actuated while the top conical or frustoconical surface is stationary.

34. The device according to any one of claims 19 to 33, wherein the bottom conical or frustoconical surface is configured to accommodate a fluid volume between at least 10 μL and 100 μL.

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