Abstract:
Microfluidic devices and methods for analyzing glycan profiles of glycoproteins are provided. Some embodiments of the devices comprise a deglycosylation column for cleaving glycans, an optional cleaning column for removing proteins, a trapping column for enriching glycans, and a separation column for resolving glycans. The devices and methods significantly improve the speed and sensitivity of glycan analysis.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    The analysis of glycoprotein structure and function, known as glycomics, has become a new and important area of research. Glycosylation is the most common post-translational modification of cell surface and extracellular matrix proteins. Glycoproteins play an important role in cell-adhesion and immune response. Changes in abundance and glycan profiles have been correlated with progression of diseases, such as cancer and rheumatoid arthritis. In addition, the analysis of glycan profiles is critical in the bio-therapeutic industry. For example, antibody bio-therapeutics contain glycosylated amino acids that assist in maintaining drug activity and preventing drug rejection by the immune system. Therefore, companies that make these bio-therapeutics have to monitor and verify their glycan profiles. 
         [0002]    Glycan analysis is usually performed by capillary electrophoresis or mass spectrometry methods. In either case, the glycans must be removed from the glycoproteins, separated and analyzed. The process of removing glycans from glycoproteins traditionally includes an in-solution enzymatic reaction with PNGase F that requires a 24-hour incubation time. After the enzyme reaction, protein precipitation is needed to separate the glycans from the proteins for analysis of the glycans. Finally, the free glycans are analyzed with mass spectrometry or capillary electrophoresis. These steps are time-consuming and cumbersome, and the manual operations are error-prone. Furthermore, a relatively large quantity of starting materials is required to generate high quality data. Therefore, a better way of performing glycan analysis is desirable. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0003]      FIG. 1A  is a schematic diagram of a top-down view of a microfluidic chip that can be used for glycan analysis of glycoproteins.  FIG. 1B  is a schematic diagram of a top-down view of a switching element in the form of a rotor that can be coupled with the chip shown in  FIG. 1A .  FIG. 1C  shows one state of a valve system formed when the chip of  FIG. 1A  is coupled with the rotor of  FIG. 1B . In this state, the trapping column is connected to the deglycosylation column.  FIG. 1D  shows another state of the valve system in which the trapping column is connected to the separation column. 
           [0004]      FIG. 2  shows two layers of a microfluidic chip. 
           [0005]      FIG. 3  shows a three-dimensional view of how deglycosylation and glycan enrichment occur in a microfluidic device of the present application. Some channels from a switching element, which is coupled with the device, are also shown. 
           [0006]      FIG. 4  shows a three-dimensional view of a microfluidic device switched to a position to send collected glycans to a separation column. Some channels from a switching element, which is coupled with the device, are also shown. 
           [0007]      FIGS. 5 and 6  illustrate a three-dimensional view of a microfluidic device that comprises three layers. Some channels from a switching element, which is coupled with the device, are also shown. 
           [0008]      FIG. 7  shows the mass spectral scan of an antibody sample that has been processed on-chip (right-hand side) or in-solution (left-hand side). 
           [0009]      FIG. 8  shows the chemical mechanism of enzymatic action of PNGase F. 
           [0010]      FIG. 9  shows the identity of the glycans corresponding to the peaks in the mass spectral scan shown in  FIG. 7 . 
           [0011]      FIG. 10  demonstrates separation of glycan isoforms G1′ and G1″ after a short gradient in liquid chromatography. 
       
    
    
     DESCRIPTION OF THE INVENTION 
       [0012]    The present invention provides a new, fast, and streamlined approach to preparing, separating and analyzing glycans using a microfluidic device. Moreover, due to the design of the microfluidic device of this invention, a higher recovery yield of glycans and superior chemical separation of isoforms are achieved, and a significantly smaller amount of starting material is required, compared to traditional methods. 
         [0013]    Prior to describing the invention in further detail, the terms used in this application are defined as follows unless otherwise indicated. 
       DEFINITION 
       [0014]    In this specification and the appended claims, the singular form “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. 
         [0015]    A “microfluidic device” is a device comprising chambers and/or channels of micron or submicron dimensions that allow passage of fluid. The chambers or channels are generally 1 μm to less than 1000 μm in diameter (or, if not circular, the largest dimension of the cross section), such as 1 μm to 500 μm, 10 μm to 300 μm, 50 μm to 250 μm, by way of examples. 
         [0016]    A “column” is an apparatus for a particular purpose, usually for preferentially binding or retaining a substance or class of substances. Typically, a column comprises a housing in which a filling is located, and the filling is capable of preferentially binding or retaining a substance or class of substances. In some embodiments, the filling may merely provide a medium for various substances to move through, while the substances move in different speeds (e.g., due to an electric field). A column (or a housing or filling) can be of any size, shape or structure, and made of any material, that is consistent with the purpose of the column. 
         [0017]    As used herein, “connect” may occur by direct or indirect connections. A direct connection means two objects that are connected have a physical contact with each other. An indirect connection means two objects that are connected do not physically contact each other, but are connected through at least one object in between. 
         [0018]    As used herein, if two objects are in “fluid communication,” there is a conduit between the two objects that allows fluid to flow from one of the objects to the other. A conduit may be a pore, orifice, opening, channel, tube, or the like. 
         [0019]    The term “carbohydrate” refers to any compound in the carbohydrate family, including sugars, disaccharides, oligosaccharides, polysaccharides, simple carbohydrates, complex carbohydrates, and the like. 
       Device and Methods 
       [0020]      FIG. 1A  illustrates an exemplary device  101  in accordance with one embodiment of the present invention. The device  101  comprises a deglycosylation column  110  for cleaving glycans from glycoprotein samples, a trapping column  120  for trapping and enriching the cleaved glycans, and a separation column  130  for separating the glycans. Although a particular shape is drawn for the device  101  and each component of the device, other variations in shape or relative size can be used for this invention. Similarly, the other devices and components described in this disclosure are not limited to the shapes or sizes shown in the drawings. 
         [0021]    The deglycosylation column  110  comprises a solid support to which an enzyme is attached, with the enzyme capable of cleaving carbohydrates from a glycoprotein. In most glycoproteins, the carbohydrate moiety is attached to the nitrogen of the amide group in asparagine residues (N-linked glycans), or the oxygen of the hydroxyl group in serine or threonine residues (O-linked glycans). Any enzyme that can cleave the carbohydrate moiety from glycoproteins can be used in the present invention, including enzymes that are specific for N-linked or O-linked glycans. These enzymes are known in the art and include, without being limited to, PNGase F, β-N-Acetyl-glucosaminidase, α-Fucosidase, β-Galactosidase, α-Galactosidase, α-Neuraminidase, α-Mannosidase, β-Glucosidase, β-Xylosidase, β-Mannosidase, Endo F 1 , Endo F 2 , Endo F 3 , and Endo H. Materials and methods of immobilizing proteins to solid supports are also known in the art (see, e.g., Palm and Novotny, 2005). For example, the solid support in the deglycosylation column  110  may be glass or polymer beads, or a monolithic medium (such as polymethacrylate, polystyrene, polyacrylamide, or the like). 
         [0022]    The trapping column  120  is capable of preferentially binding carbohydrates but not proteins. For example, hydrophilic interaction liquid chromatography (HILIC) stationary phase can be used to retain and desalt the glycans released from glycoproteins in the deglycosylation column. The separation column  130  is capable of separating glycans based on their physical and/or chemical properties. Two main categories of the separation column are liquid chromatography columns and capillary electrophoresis columns. 
         [0023]    The device  101  also contains a plurality of inlet/outlet ports  141 - 146 . An inlet/outlet port (or “port”) can be a hole, orifice, opening, any of the above connected to a conduit (especially a short conduit), or the like, as long as the port allows fluid to pass from one end of the port to the other. The ports  141 - 146  can be used to connect different parts of the device at different stages when device  101  is aligned with and coupled to appropriate channels. For example, device  101  can be fit on top of a rotor  150  that comprises three channels  151 ,  153  and  155  ( FIG. 1B ). The rotor  150  can be rotated so that different ports in device  101  are connected by each channel in the rotor when the rotor is in a different position. For example, the rotor can be rotated to a position wherein inlet/outlet ports  141  and  142  are connected by channel  151  ( FIG. 1C ). At this position, inlet/outlet ports  143  and  144  are also connected (by channel  153 ), and so are inlet/outlet ports  145  and  146  (by channel  155 ). A sample that contains, or is suspected to contain, glycoproteins is loaded into the deglycosylation column  110  through an inlet port  111 . The glycoproteins are digested in the deglycosylation column  110 , releasing free glycans as well as the remaining part of the glycoproteins. This mixture flows to the end of the deglycosylation column, which is connected to port  141 , and enters the trapping column  120  via channel  151 . In the trapping column  120 , the glycans are bound to the carbohydrate-binding substance, while the remaining components of the mixture flow through the column and are collected or discarded via channel  155 . 
         [0024]    The system is then switched to a different position (see  FIG. 1D ), in which the following inlets/outlets are connected: port  142  with port  143 , port  144  with port  145 , and port  146  with port  141 . Thus, the trapping column  120  and the separation column  130  are now in fluid communication through channel  153 , and the deglycosylation column  110  is no longer connected with the trapping column  120 . An elution solution can be added to the trapping column  120  from port  143 , and the eluate flows from port  145  to port  144  and enters the separation column  130 . The glycans are then separated in the separation column  130 . The outlet of the separation column  130  can be connected to the sample inlet of a mass spectrometer to further analyze the glycans by mass spectrometry. 
         [0025]    Although a rotor is described above as a switching element to change the fluid communication state of the columns in device  101 , other switching elements can be utilized. For instance, a set of channels and valves can be engaged with device  101  so that, upon switching one valve or multiple valves, the deglycosylation column is disconnected from the trapping column, and the trapping column is connected with the separation column. 
         [0026]    In some embodiments, the deglycosylation column  110  and the trapping column  120  are continuous. For example, instead of two discontinuous columns  110  and  120 , device  101  can have a chamber that comprise a deglycosylation material (a deglycosylation enzyme immobilized to a solid support) in a first part of the chamber to constitute a deglycosylation column, and a trapping material (which preferentially binds carbohydrates but not proteins) in the next part of the chamber to form a trapping column. As another example, a deglycosylation column can be connected with a trapping column by an orifice or conduit in device  101 , so the two columns are in fluid communication within the structure of device  101 . For these embodiments, a simpler switching element can be used, which does not have to connect the deglycosylation column with the trapping column. It is conceivable that the switching element may comprise a minimum of a channel and a valve controlling the channel, wherein the channel can be positioned to connect the trapping column and the separation column. When the valve is switched off, the trapping column is not in fluid communication with the separation column. In this state, sample loading, deglycosylation, and glycan enrichment (trapping) can be performed. The valve is then switched on to allow fluid communication between the trapping column and the separation column for eluting the trapping column and separating the glycans. 
         [0027]    In some embodiments, different parts of the device are located on different layers or chips, which are assembled to form the device (see, e.g., U.S. Patent Publication No. 2006/0171855). For example,  FIG. 2  shows a chip  210  containing the trapping column  120  and the separation column  130 . Another chip (“deglycosylation chip”)  220  contains the deglycosylation column. The deglycosylation chip  220  can be connected to the chip  210  by, for example, mechanically sealing on top of chip  210 , in a manner that allows fluidic communication between inlet/outlet port  141  of chip  210  with port  141 ′ of deglycosylation chip  220 . Note that the deglycosylation chip  220  does not contain counterparts of inlet/outlet ports  144  and  145  in this design, because  144  and  145  are used for communication between the trapping column and the separation column, both of which are on chip  210 . Chip  220 , however, may have counterparts of inlet ports  143  and  146  ( 143 ′ and  146 ′) for alignment purposes only. Thus, port  143  is aligned with port  143 ′, and port  146  is aligned with port  146 ′ when chips  210  and  220  are assembled. 
         [0028]    In operation, the assembled, two-chip device is engaged with a switching element that contains multiple channels, such as the rotor shown in  FIG. 1B . A sample is loaded into inlet port  111 , which is the inlet to the deglycosylation column  110 . The reaction mixture after deglycosylation flows, sequentially, to inlet/outlet port  141 , inlet/outlet port  141 ′ on chip  210  which is connected to port  141 , a channel in the rotor that connects port  141 ′ with port  142 , port  142 , and trapping column  120 . After trapping, the rotor is switched to allow fluid communication between trapping column  120  and the separation column  130  (through a channel in the rotor that connects port  144  and port  145 ), and the glycans are eluted and separated as described above. 
         [0029]      FIGS. 3 and 4  show three-dimensional views of how some embodiments of two-layer devices work in conjunction with a switching element. As illustrated in  FIG. 3 , the sample is injected into inlet port  111  to enter the deglycosylation column, which is in the shape of a loop in the embodiment in this figure. Only the beginning part and the ending part of the loop are shown. This particular embodiment differs from the one shown in  FIG. 2  in that port  141  is below inlet port  111 , which is a variation that serves the same function. The output from the deglycosylation column is led into the trapping column through channel  151 , and glycan enrichment occurs in the trapping column, which is in a lower layer compared to the deglycosylation column. Proteins without a carbohydrate moiety flow through the trapping column and exit through channel  155 . In  FIG. 4 , the trapping column is connected with channels  151  and  153  (by switching a switching element not completely shown in the figure), an elution solution is applied to the trapping column through channel  151 , and glycans are eluted from the trapping column and enters the separation column through channel  153 . 
         [0030]    In some embodiments, the device further comprises a cleaning column that is capable of preferentially binding proteins but not carbohydrates. For example, the C18, C8, or other reverse phase material retains protein while passing free glycans. For proteins such as antibodies, protein A can be used to bind the antibody but not the glycans. The proteins bound to the cleaning column can be discarded or analyzed to understand the protein profile and content in the sample. These embodiments have inlet/outlet ports that, when coupled with a switching element, allow for connection between the deglycosylation column and the cleaning column, and between the cleaning column and the trapping column, either at the same time or not. It is also contemplated that in some embodiments, the deglycosylation column can be connected with the trapping column, and the trapping column with the cleaning column, either at the same time or not. Thus, in some embodiments, all of these three columns can be connected at the same time in the order of deglycosylation-cleaning-trapping, or deglycosylation-trapping-cleaning. 
         [0031]      FIGS. 5 and 6  illustrate a three-dimensional view of a device that comprises a cleaning column in a three-layer design. The first layer includes a deglycosylation column  110 , a protein cleaning column  115  is in the middle layer, and the bottom layer contains a trapping column  120  and a separation column  130 . The figures also show three channels from a switching element. 
         [0032]    Example 1 describes experiments in which the embodiment depicted in  FIGS. 5 and 6  was used to analyze glycan compositions in an antibody sample. The results demonstrate that the device of the present invention enables a fast analysis, and it is possible to preserve the original chemical structures of the glycans in the fast analysis. In contrast, a traditional in-solution analysis performed in parallel indicates that chemical structures of the glycans changed during the prolonged incubation of the traditional analysis. Unexpectedly, isoforms of the glycans, now in their original structures (amino glycan forms), can be resolved much more readily in the separation column, whereas it had been necessary to employ a long and more time-consuming and thus expensive separation process to separate the isoforms, a problem that had been troublesome in the field. 
         [0033]    The speed of the present methods depends on complexity of the samples, as longer separation is required when more glycans need to be separated. In addition, if finer separation is desired, such as separation of isomers, a longer separation column and thus longer separation time is needed as well. By the present methods, the process of deglycosylation, cleaning, trapping and separation can generally be achieved in one minute to an hour, such as in 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the process is performed under conditions that allow a substantial amount of the glycans to remain in amino glycan forms. This substantial amount may be, for example, at least 95, 90, 80, 70, 60, 50, 40, or 30% of the glycans in the sample. 
         [0034]    The results also indicate that a small amount of sample (100 ng glycoproteins) yielded strong signals, suggesting that the amount of the sample can be reduced. Thus, in some embodiments, the present methods can be used to analyze a sample containing up to 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 ng of glycoproteins. 
         [0035]    Thus, some embodiments of the present invention provide devices, usually in the form of chips, that comprise: 
         [0036]    a deglycosylation column comprising a solid support and an enzyme immobilized to the solid support, wherein the enzyme is capable of cleaving carbohydrates from glycoproteins; 
         [0037]    a trapping column capable of binding carbohydrate; 
         [0038]    a separation column capable of separating carbohydrates; and 
         [0039]    a plurality of inlet/outlet ports; 
         [0040]    wherein said ports are configured so that when said device is aligned with a switching element that comprises a channel or a plurality of channels, the combination of said ports, columns and channel(s) forms a valve system that can be switched between at least a first state and a second state, the first state allowing fluid communication between the deglycosylation column and the trapping column, and the second state allowing fluid communication between the trapping column and the separation column. 
         [0041]    The device can optionally further comprise a cleaning column that is capable of binding proteins. The cleaning column may be used to bind and discard proteins, which are undesirable in glycan analyses. Alternatively, the cleaning column can be used to collect proteins for a protein analysis in addition to the glycan analysis. 
         [0042]    In some other embodiments, the device may comprise a deglycosylation column, a cleaning column, and a separation column, without the trapping column. These embodiments fall into two groups. One group can be used for protein analyses, in which case the separation column is one for separation of proteins rather than carbohydrates. A switching element capable of connecting the cleaning column with the separation column is to be used in conjunction with this group. The other group can be used for a shortened glycan analysis process, in which glycan enrichment is omitted. Embodiments in this group comprise a separation column for separating carbohydrates. 
         [0043]    Microfluidic devices are typically fabricated by generating channels and chambers in a material that forms the device or layers of the device. Suitable materials for forming the device, or layers of the device, are selected with regard to physical and chemical characteristics that are desirable for proper functioning of the microfluidic device. For example, a device is fabricated from a material that enables formation of high definition features, i.e., microchannels, microchambers and the like, that are of micron or submicron dimensions. Thus, the material must be capable of microfabrication using, e.g., dry etching, wet etching, laser etching, laser ablation, molding, embossing, or the like, so as to have desired miniaturized surface features. Microstructures can also be formed on the surface of a material by adding material thereto, for example, polymer channels can be formed on the surface of a glass substrate using photo-imageable polyimide. Also, all device materials used should be chemically inert and physically stable with respect to any substance with which they comes into contact when used to introduce a fluid sample (e.g., with respect to pH, electric fields, etc.). Suitable materials for forming the present devices include, but are not limited to, polymeric materials, ceramics (including aluminum oxide and the like), glass, metals, composites, and laminates thereof. 
         [0044]    Polymeric materials are typically organic polymers that are homopolymers or copolymers, naturally occurring or synthetic, crosslinked or uncrosslinked. Specific polymers of interest include, but are not limited to, polyimides, polycarbonates, polyesters, polyamides, polyethers, polyurethanes, polyfluorocarbons, polystyrenes, poly(acrylonitrile-butadiene-styrene) (ABS), acrylate and acrylic acid polymers such as polymethyl methacrylate, and other substituted and unsubstituted polyolefins, and copolymers thereof. Polyimide is of particular interest and has proven to be a highly desirable substrate material in a number of contexts. Polyetheretherketones (PEEK) also exhibit desirable biofouling resistant properties. 
         [0045]    The devices of the invention may also be fabricated from a “composite,” i.e., a composition comprised of unlike materials. The composite may be a block composite, e.g., an A-B-A block composite, an A-B-C block composite, or the like. Alternatively, the composite may be a heterogeneous combination of materials, i.e., in which the materials are distinct from separate phases, or a homogeneous combination of unlike materials. As used herein, the term “composite” is used to include a “laminate” composite. A “laminate” refers to a composite material formed from several different bonded layers of identical or different materials. Other preferred composite materials include polymer laminates, polymer-metal laminates, e.g., polymer coated with copper, a ceramic-in-metal or a polymer-in-metal composite. One preferred composite material is a polyimide laminate formed from a first layer of polyimide such as Kapton®, that has been co-extruded with a second, thin layer of a thermal adhesive form of polyimide known as KJ®, both available from DuPont (Wilmington, Del.). 
         [0046]    The present devices can be fabricated using any convenient method, including, but not limited to, micromolding and casting techniques, embossing methods, surface micro-machining and bulk-micromachining. The latter technique involves formation of microstructures by etching directly into a bulk material, typically using wet chemical etching or reactive ion etching (“RIE”). Surface micro-machining involves fabrication from films deposited on the surface of a substrate. An exemplary surface micro-machining process is known as “LIGA.” See, for example, Becker et al. (1986), Ehrfeld et al. (1988), and Guckel et al. (1991). LIGA involves deposition of a relatively thick layer of an X-ray resist on a substrate followed by exposure to high-energy X-ray radiation through an X-ray mask, and removal of the irradiated resist portions using a chemical developer. 
         [0047]    Another technique for preparing the present devices is laser ablation. In laser ablation, short pulses of intense ultraviolet light are absorbed in a thin surface layer of material. Preferred pulse energies are greater than about 100 millijoules per square centimeter and pulse durations are shorter than about 1 microsecond. Under these conditions, the intense ultraviolet light photo-dissociates the chemical bonds in the substrate surface. The absorbed ultraviolet energy is concentrated in such a small volume of material that it rapidly heats the dissociated fragments and ejects them away from the substrate surface. Because these processes occur so quickly, there is no time for heat to propagate to the surrounding material. As a result, the surrounding region is not melted or otherwise damaged, and the perimeter of ablated features can replicate the shape of the incident optical beam with precision on the scale of about one micron or less. Laser ablation will typically involve use of a high-energy photon laser such as an excimer laser of the F 2 , ArF, KrCl, KrF, or XeC1 type. However, other ultraviolet light sources with substantially the same optical wavelengths and energy densities may be used as well. Laser ablation techniques are described, for example, by Znotins et al. (1987), and in U.S. Pat. Nos. 5,291,226 and 5,305,015 to Schantz et al. 
         [0048]    Another aspect of this invention provides systems that comprise a device of the present invention. The system may further comprise a switching element for switching a valve system to allow selective connections between the various columns of the device. The switching element may be, for example, a rotor comprising a plurality of channels that can be aligned with the inlets/outlet ports of the chip when the chip is engaged with the rotor. Furthermore, the rotor can be switched between at least two different states, each of which allows for connection of different ports in the chip. The switching element may also be a sliding element that can be slid between at least two different positions to connect different ports, such as those described in U.S. Pat. No. 6,702,256. Other switching mechanisms can also be used. The system may optionally comprise an interface for transmitting the output of the device (separated glycans) to the inlet of a mass spectrometer or other analysis tools. Alternatively, the output of the device may directly enter the ionization area of a mass spectrometer. The system may further comprise a mass spectrometer or other analysis tools. 
         [0049]    Other embodiments also provide kits that comprise a device of the present invention. The kit can further comprise any one or any combination of the following: sample solution, elution solution for any of the columns, reconstitution reagents for any of the columns, a standard sample (such as an antibody standard, glycan standard, etc.), instructions for use, and a container for the components. 
         [0050]    The following examples are offered to illustrate various embodiments of this invention and are not to be construed in any way as limiting the scope of the present invention. While certain embodiments of this invention are particularly shown and described, various changes in form and details may be made therein without departing from the spirit and scope of the invention. 
       EXAMPLES 
       [0051]    In the examples below, the following abbreviations have the following meanings. Abbreviations not defined have their generally accepted meanings. 
         [0052]    ° C.=degree Celsius 
         [0053]    hr=hour 
         [0054]    min=minute 
         [0055]    sec=second 
         [0056]    mM=millimolar 
         [0057]    μM=micromolar 
         [0058]    nM=nanomolar 
         [0059]    μm=micrometer 
         [0060]    ml=milliliter 
         [0061]    μl=microliter 
         [0062]    nl=nanoliter 
         [0063]    mg=milligram 
         [0064]    μg=microgram 
         [0065]    HPLC=high performance liquid chromatography 
         [0066]    LC=liquid chromatography 
         [0067]    MS=mass spectrometry 
         [0068]    QTOF=quadrupole-time of flight 
       Example 1 
     Glycan Analysis with on-Chip Enzymatic Reactions 
     Chip Fabrication 
       [0069]    PNGase F enzyme was chemically attached to epoxide-modified 5 μm silica beads in phosphate buffer (pH 8) over 22 hr. The beads were then packed into a chamber located in a first polyimide chip. Reverse-phase 5-μm C8 beads were packed into a second chamber on a second polyimide chip for protein cleaning. The third chip layer contained a sample enrichment column (40 nl), a separation column (75 mm) filled with graphitized carbon for HPLC analysis, and an integrated MS tip for input into a mass spectrometer. All three chips were stacked, aligned and inserted into a chip frame holder. 
       Methods 
       [0070]    The antibody samples were diluted in phosphate buffer to a concentration of 6 pmol/μl in phosphate buffer (pH 7.5). The HPLC system operated using typical mobile phases of 0.1% formic acid in MS-grade water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B). The autosampler was set to inject 0.1 μl of antibody. The capillary pump was set to pass the sample through the deglycosylation column and the C8 column at a flow rate of 1 μl/min. This flow rate corresponds to a deglycosylation time of 8 seconds, and resulted in 100% deglycosylation. The solvent used for loading and deglycosylation was 50 mM sodium phosphate. The gradient for fast deglycosylation and glycan analysis was 2-32% Solvent B in 1.5 min, 32-75% Solvent B in 30 sec and held at 75% Solvent B for another 30 sec, followed by 75-2% Solvent B over the last 30 sec. with a nano pump flow of 300 nl/min. A longer gradient was used to separate isomers. 
         [0071]    The separated glycans were analyzed using a QTOF tandem mass spectrometer with an electrospray ionization source. The mass spectra were recorded in positive ion mode. Voltage of the cap was set to 1750V, with a drying gas of 325° C. at 8 L/min. The fragmentation voltage was 120V. The skimmer voltage was 65V. Data was acquired in 2 GHz mode. 
       Results 
       [0072]    Antibody samples were deglycosylated and the resulting glycans were separated by HPLC and analyzed by mass spectrometry, using the chip and method as described above. The expected glycans include any of the following: 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Candidate glycans 
               
             
          
           
               
                   
                   
                   
                 Main ion 
               
               
                   
                   
                 Glycan 
                 species 
               
               
                 Name 
                 Structure 
                 Mass 
                 [m/z + 2] + 2 
               
               
                   
               
             
          
           
               
                 G0 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 1462.54 
                 732.27 
               
               
                   
               
               
                 G1′ 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 1624.59 
                 813.29 
               
               
                   
               
               
                 G1″ 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
                   
               
               
                   
               
               
                 G2 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 1786.65 
                 894.32 
               
               
                   
               
               
                 Mann-5 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 1234.43 
                 618.21 
               
               
                   
               
               
                 Open circle: galactose 
               
               
                 Square: GlcNAc 
               
               
                 Closed circle: Mannose 
               
               
                 Triangle: Fucose 
               
             
          
         
       
     
         [0073]      FIG. 7  (left-hand side) shows a typical result from the traditional, in-solution analysis in which the sample was cleaved by PNGase F in solution overnight in a protocol that lasted 24 hours in total. Of the major peaks,  303  and  305  correspond to two stereoisomers (α- and β-anomers) of G0, and  304  and  306  correspond to the two isomers of G1, G1′ and G1″ (see Table 1 above). All these glycan species eluted around 3.4-3.7 minutes. In addition, there are also minor peaks  301  and  302  eluting around 3.2 minutes. However, the results of the on-chip analysis using the same sample were different. As shown in  FIG. 7 , right-hand side, the sample yielded a prominent peak ( 310 ) and two smaller peaks ( 311  and  312 ), all around 3.2 minutes. Four minor peaks  313 - 316  appeared between 3.4 and 3.7 minutes, where the major peaks in the in-solution analysis were. In fact, the positions of  313 - 316  were in good agreement with peaks  303 - 306  in the in-solution analysis, just like peaks  310  and  311  eluted at about the same times as peaks  301  and  302 . 
         [0074]    The discrepancy between the in-solution and on-chip results can be explained in view of the mechanism of action of PNGase F and the efficient, on-line nature of the chip. According to Rasmussen et al., 1992 (see FIG. 8), PNGase F acts by first cleaving the C—N bond of the glycosylated asparagine side chain, and the asparagine residue is converted to aspartic acid. The cleaved glycan is an amino glycan initially, but the amino group is slowly hydrolyzed to a hydroxyl group with the release of ammonia. Taking into account the masses of each peak in  FIG. 7 , we postulated that peak  310  corresponds to amino glycan of G0, which is hydrolyzed to hydroxyl glycans (peaks  313  and  315 ) upon a long incubation. To test this hypothesis, a time-course experiment was conducted in which the sample was allowed to stay in the deglycosylation column in our chip for 0, 15, 30, 60, 120 and 240 minutes, respectively. Indeed, peak  310  decreased and peaks  313  and  315  increased in intensity with each prolonged incubation time. Similarly, the peaks for G1 in the in-solution analysis ( 304  and  306 ) also decreased over time, and  311  increased. Thus, based on the time-dependent changes and masses of the peaks, we conclude (see  FIG. 9 ) that peaks  310 ,  311  and  312  correspond to amino glycans, and peaks  313 - 316  correspond to hydroxyl glycans. There are two peaks ( 313  and  315 , and  314  and  316 ) for each hydroxyl glycan, because hydroxyl glycans isomerize easily between the α-anomer and the β-anomer. 
         [0075]    The on-line nature of the chip also contributes to efficiency of the reaction. When a sample enters the deglycosylation column and moves along, each “front” of the sample comes in contact with an excess amount of the enzyme and is cleaved instantly. On the other hand, the in-solution incubation mixture contains a lower enzyme to substrate ratio for each substrate, and the reaction tends to leave a population of various incompletely digested products. Furthermore, in the on-line process, the cleaved glycans continue to move along, bind to the trapping column, and become separated in the separation column. Thus, the glycans are bound by other molecules most of the time and have less chance to isomerize or undergo other chemical reactions. 
         [0076]    The ability of the present invention to catch the amino glycans makes it possible to separate the two isoforms of G1 efficiently. G1 exists as two isomers (G1′ and G1″ in Table 1). It was believed that G1′ and G1″ could only be separated using a very long gradient. Therefore, it has been a problem to accurately analyze glycan compositions without spending the resources and time for a long separation step. We discovered, however, that the amino forms can be readily separated. The following short gradient was used to run the separation column in the chip described in this example, and G1′ and G1″ were clearly resolved ( FIG. 10 ) in a matter of minutes: 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 Time (min) 
                 % Solvent B 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 0 
                 2 
               
               
                   
                 10 
                 22 
               
               
                   
                 12 
                 80 
               
               
                   
                 15 
                 80 
               
               
                   
                 16 
                 2 
               
               
                   
                   
               
             
          
         
       
     
         [0077]    Thus, these results demonstrate that the chips described in this disclosure not only significantly shorten the time required for glycan analysis, but the data obtained from the chips reflect the structures of the glycans more faithfully. In addition, only a small amount of initial sample is required for the chip to achieve the same results. The sample in this example contained only 100 ng of glycoproteins, and the signals in mass spectra were very high. Therefore, a lower amount of sample can be used. 
       Exemplary Embodiments 
       [0078]    Embodiments of the present invention include, without being limited to, the following: 
         [0000]    A. A microfluidic device for removing carbohydrate from a glycoprotein, comprising: 
         [0079]    a deglycosylation column comprising a solid support and an enzyme immobilized to the solid support, wherein the enzyme is capable of cleaving carbohydrates from glycoproteins; 
         [0080]    a trapping column that is capable of binding carbohydrates; 
         [0081]    a separation column capable of separating carbohydrates; and 
         [0082]    a plurality of inlet/outlet ports; 
         [0000]    wherein said ports are configured so that when said device is coupled with a switching element that comprises at least one channel, the combination of said ports, columns and at least one channel forms a valve system that can be switched between at least a first state and a second state, the first state allowing fluid communication between the deglycosylation column and the trapping column, and the second state allowing fluid communication between the trapping column and the separation column.
 
B. The device of embodiment A, further comprising a cleaning column capable of binding proteins, wherein the cleaning column is configured to be connectable to the deglycosylation column and/or the trapping column by the valve system.
 
C. The device of embodiment A or B, wherein the enzyme is N-glycosidase F.
 
D. The device of any one of embodiments A-C, wherein the solid support in the deglycosylation column comprises beads or a monolithic medium.
 
E. The device of any one of embodiments A-D, wherein the separation column is a liquid chromatography column.
 
F. The device of any one of embodiments A-D, wherein the separation column is a capillary electrophoresis apparatus.
 
G. The device of any one of embodiments A-F that comprises two layers, wherein the deglycosylation column is in one layer, and the trapping column and separation column are in the other layer.
 
H. The device of any one of embodiments B-F that comprises three layers, wherein the deglycosylation column is in a first layer, the cleaning column is in a second layer, and the trapping column and separation column are in a third layer.
 
I. A system for analyzing a sample, comprising the device of any one of embodiments A-H, the switching element, and a mass spectrometer.
 
J. The system of embodiment I, wherein the mass spectrometer comprises an electrospray ion source.
 
K. A method for analyzing the carbohydrate moieties of glycoproteins, comprising:
 
         [0083]    applying a sample that may comprise glycoproteins to the device of embodiment A; 
         [0084]    digesting the glycoproteins in the deglycosylation column to result in cleaved carbohydrates; 
         [0085]    binding the cleaved carbohydrates to the trapping column; 
         [0086]    eluting the cleaved carbohydrates from the trapping column; and 
         [0087]    separating the cleaved carbohydrates with the separation column. 
         [0000]    L. The method of embodiment K, further comprising removing proteins after the digesting with a cleaning column capable of binding proteins.
 
M. The method of embodiment K or L, wherein the cleaved carbohydrates are separated by liquid chromatography.
 
N. The method of embodiment K or L, wherein the cleaved carbohydrates are separated by capillary electrophoresis.
 
O. The method of any one of embodiments K-N, further comprising analyzing the cleaved carbohydrates using mass spectrometry.
 
P. The method of any one of embodiments K-O, wherein the sample contains up to 50 ng of glycoproteins.
 
Q. The method of any one of embodiments K-P, wherein the method is completed within 10 minutes.
 
R. The method of any one of embodiments K-Q that is performed under conditions that allow at least some of the cleaved carbohydrates to remain in amino glycan forms. Preferably, a substantial amount of the cleaved carbohydrates remain in amino glycan forms.
 
S. The method of any one of embodiments K-R, wherein the glycoproteins are digested with N-glycosidase F.
 
T. A kit for glycan analysis, comprising the device of any one of embodiments A-H and at least one reagent for sample dilution or column elution.
 
       REFERENCES 
       [0000]    
       
         Palm and Novotny (2005), “A monolithic PNGase F enzyme microreactor enabling glycan mass mapping of glycoproteins by mass spectrometry,” Rapid Comm. Mass Spectrometry 19: 1730-1738. 
         Rasmussen et al. (1992), “Identification and derivatization of (oligosaccharyl)amines obtained by treatment of asparagine-linked glycopeptides with N-GLYCANASE enzyme,” J. Am. Chem. Soc., 114(3): 1124-1126. 
         Becker et al. (1986), “Fabrication of Microstructures with High Aspect Ratios and Great Structural Heights by Synchrotron Radiation Lithography Galvanoforming, and Plastic Moulding (LIGA Process),” Microelectronic Engineering 4(1):35-36. 
         Ehrfeld et al. (1988), “1988 LIGA Process: Sensor Construction Techniques via X-Ray Lithography,” Tech. Digest from IEEE Solid-State Sensor and Actuator Workshop, Hilton Head, S.C. 
         Guckel et al. (1991), J. Micromech. Microeng. 1: 135-138. 
         Znotins et al. (1987), Laser Focus Electro Optics, at pp. 54-70. 
         U.S. Pat. Nos. 6,702,256; 5,291,226 and 5,305,015. 
         U.S. Patent Publication No. 2006/0171855. 
       
     
         [0096]    All of the publications, patents and patent applications cited above or elsewhere in this application are herein incorporated by reference in their entirety to the same extent as if the disclosure of each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety. 
         [0097]    A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention.