Systems and methods for preparing samples and performing real-time assays on those samples

Abstract

To provide systems and methods that facilitate the automatic preparation of a sample of a product containing molecules for analysis and automatic performance of an assay of that sample.SOLUTION: A method includes the steps of: moving a sample containing molecules from a first vial to a sample loop; moving the sample from the sample loop to a first multi-port valve; moving the sample from the first multi-port valve to a second multi-port valve; moving the sample from the second multi-port valve to a capture column; capturing the molecules in the sample in the capture column, thereby separating the molecules in the sample from a matrix of the sample; moving an elution buffer solution to the capture column, thereby eluting molecules captured by the capture column and moving an elution / molecule mixture to a second vial, the second vial comprising a flow-through vial; and moving the molecules in the second vial to an analytical device for analysis of the molecules.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 805,902, filed February 12, 2019, and U.S. Provisional Patent Application No. 62 / 951,346, filed December 20, 2019, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates generally to assays, and more particularly to systems and methods for preparing samples and performing real-time assays on those samples.

[0003] Sequence Listing This application is filed with an electronic Sequence Listing. The Sequence Listing, submitted as a file entitled "53661_Seqlisting.txt," was created on February 11, 2020, and is 263,959 bytes in size. The information in the electronic Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0004] Assays are generally performed to quantify one or more attributes of an analyte, such as a drug, biochemical, or cell. One example of such an assay is a multi-attribute method (MAM) assay, which can detect and quantify a sample's critical quality attributes (CQAs) (identified by a target product quality profile (QTPP)). (Development of a quantitative mass spectrometry multi-attribute method for characterization, quality control testing, and disposition of biologics. Rogers RS, Nightlinger NS, Livingston B, Campbell P, Bailey R, Balland A. MAbs. 2015;7(5):881-90). MAM assays are manually operated processes performed, for example, in a macromolecular release test (LMRT) laboratory. MAM is a liquid chromatography (LC)-mass spectrometry (MS)-based peptide mapping method that involves three steps: (1) sample preparation (which may include, for example, polypeptide denaturation, reduction, alkylation, and digestion), (2) separation of digested polypeptides by LC and their detection by MS, and (3) analysis of the data for CQAs of interest and detection of new signals (i.e., peaks) when compared to reference standards.

[0005] CQAs are chemical, physical, or biological properties that exist within a specific value or range of values. For example, in polypeptide therapeutic macromolecules, physical attributes and modifications of amino acids (the building blocks of the polypeptide) are important CQAs that are monitored during and after manufacturing and during drug development. Unlike traditional analytical assays that track changes in peak size and shape of the entire polypeptide or portions thereof, MAMs detect specific CQAs at the amino acid level.

[0006] Analysis of the glycosylation profile of therapeutic polypeptides is often a critical quality assurance (CQA). This is especially true for biosimilar products, whose glycosylation profile must be comparable to that of their reference product. Known processes for performing glycosylation assays require product samples to be manually collected, delivered to a laboratory, and manually concentrated, purified, and prepared for analysis. These known, manual processes typically require approximately five days. This time lag increases costs and delays drug (reference and biosimilar) development and ultimately drug release. During drug development, for example, a five-day delay when optimizing culture conditions for optimal glycosylation can accumulate and prevent important, novel polypeptide therapeutics from reaching patients. Furthermore, such delays can result in profile determinations being made post-manufacturing, leading to the remanufacturing of products that do not meet specifications, as opposed to adjusting manufacturing parameters in real time. Therefore, there is a need for efficient and more rapid methods to facilitate CQA analysis, including sample preparation for such analyses. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Development of a quantitative mass spectrometry multi-attribute method for characterization, quality control testing and disposition of biologics.Rogers RS,Nightlinger NS,Livingston B,Campbell P,Bailey R,Balland A.MAbs.2015;7(5):881-90 Summary of the Invention [Means for solving the problem]

[0008] One aspect of the present disclosure includes a method for detecting a molecule in a sample, the method comprising: (a) transferring a sample containing the molecule from a first vial to a sample loop; (b) transferring a quantity of the sample from the sample loop to a first multiport valve; (c) transferring a quantity of the sample from the first multiport valve to a second multiport valve fluidly coupled to and positioned downstream of the first multiport valve; (d) transferring a quantity of the sample from the second multiport valve to a trap column when the second multiport valve is in a first position; and (e) transferring a quantity of the sample from the second multiport valve to a trap column. (f) transferring an elution buffer solution from a buffer source to the capture column, thereby eluting the molecules captured by the capture column, and transferring an elution / molecule mixture comprising the elution buffer solution and the eluted molecules to a second vial positioned downstream of the capture column, wherein the second vial comprises a flow-through vial; and (g) transferring the molecules in the second vial to an analytical device for analysis of the molecules.

[0009] Another aspect of the present disclosure provides a method for detecting polypeptides in a sample, the method comprising: (a) transferring a sample comprising a polypeptide from a first vial to a sample loop; (b) transferring a quantity of the sample from the sample loop to a first multiport valve; (c) transferring a quantity of the sample from the first multiport valve to a second multiport valve fluidly coupled to and positioned downstream of the first multiport valve; (d) transferring a quantity of the sample from the second multiport valve to a polypeptide binding column when the second multiport valve is in a first position; and (e) isolating the polypeptides in the sample in the polypeptide binding column. (f) transferring an elution buffer solution from a buffer source to the polypeptide binding column, thereby eluting the polypeptides captured by the polypeptide binding column and transferring the elution / molecular mixture comprising the elution buffer solution and the eluted polypeptides to a second vial disposed downstream of the polypeptide binding column, the second vial comprising a flow-through vial; and (g) transferring the polypeptides in the second vial to an analytical device for analysis of the polypeptides. Note that the "vial" or "first vial" to which the sample is initially transferred, as used herein, can comprise or consist of any suitable vial, such as a flow-through vial or a non-flow-through vial. Furthermore, non-flow-through vials (and flow-through vials) can be used in the methods and systems described herein as temporary or transition containers for intermediate / partially processed samples before they are transported to the next processing step in the system.

[0010] Another aspect of the present disclosure provides a method for analysing a polypeptide comprising: (a) transferring a sample comprising a polypeptide from a first vial to a sample loop; (b) transferring a quantity of the sample from the sample loop to a first multiport valve; (c) transferring a quantity of the sample from the first multiport valve to a second multiport valve fluidly coupled to and positioned downstream of the first multiport valve; (d) transferring a quantity of the sample from the second multiport valve to a polypeptide binding column; (e) binding the polypeptides in the quantity of the sample to the polypeptide binding column, thereby separating the polypeptides in the sample from the sample matrix; (f) transferring a glycosidase (such as a glucanase) to the polypeptide binding column through the second multiport valve to release glycans from the bound polypeptides; (g) transferring the released glycans from the polypeptide binding column to a second vial disposed downstream of the polypeptide binding column, the second vial comprising a flow-through vial; (h) mixing the released glycans with a glycan labeling reagent in the second vial; (i) transferring the mixture of the released glycans and the glycan labeling reagent to a reaction coil through a first multiport valve; (j) incubating the mixture of the released glycans and the glycan labeling reagent in the reaction coil, thereby labeling the glycans; (k) transferring the mixture from the reaction coil to a third vial, the third vial also comprising a flow-through vial; and (l) transferring the labeled glycans in the third vial to an analytical device for analysis of the labeled glycans. The present invention provides a method comprising:

[0011] Another aspect of the present disclosure provides a closed system including a first vial adapted to contain a sample containing a molecule, one or more altering agents, a sample loop adapted to receive the sample from the first vial, a first multiport valve fluidly connected to the sample loop and configured to obtain a volume of the sample through a first port of the first multiport valve, and a second multiport valve fluidly connected to the first multiport valve and positioned downstream of the first multiport valve. The closed system includes a trapping column positioned to be fluidly connected to the second multiport valve when the second multiport valve is in a first position. The trapping column is configured to capture molecules from the volume of the sample. The closed system also includes a desalting column positioned to be fluidly connected to the second multiport valve when the second multiport valve is in a second position different from the first position. The desalting column is configured to reduce the salt concentration of the molecule. The closed system further includes a first reaction coil positioned to be selectively fluidly connected to the first multiport valve. The first reaction coil is configured to receive one or more modifying agents and to incubate the molecules and the one or more modifying agents to modify the molecules. The closed system further includes a second vial positioned downstream of the second multiport valve and fluidly connected to the second multiport valve. The second vial is configured to receive a mixture containing the molecules from the capture column, the desalting column, or the first reaction coil. The second vial includes a flow-through vial. In some embodiments, the flow-through vial is configured to separate the molecules from the remainder of the mixture so that the molecules can be analyzed.

[0012] Another aspect of the present disclosure provides a closed system including a first vial adapted to contain a sample containing a polypeptide, one or more altering agents, a sample loop adapted to receive the sample from the first vial, a first multiport valve fluidly connected to the sample loop and configured to obtain a volume of the sample through a first port of the first multiport valve, a second multiport valve fluidly connected to the first multiport valve and positioned downstream of the first multiport valve, and a capture column configured to be fluidly connected to the second multiport valve when the second multiport valve is in a first position. The capture column is configured to capture the polypeptide from the volume of the sample. The closed system also includes a desalting column fluidly connected to the second multiport valve when the second multiport valve is in a second position different from the first position. The desalting column is configured to reduce the salt concentration of the polypeptide. The closed system further includes a first reaction coil configured to be selectively fluidly connected to the first multiport valve. The first reaction coil is configured to receive one or more modifying agents and is configured to incubate the polypeptide and the one or more modifying agents to modify the polypeptide. The closed system further includes a second vial positioned downstream of the second multiport valve and fluidly connected to the second multiport valve. The second vial is configured to receive a mixture containing the polypeptide from the capture column, desalting column, or first reaction coil. The second vial includes a flow-through vial configured to separate the polypeptide from the remainder of the mixture so that the polypeptide can be analyzed.

[0013] Another aspect of the present disclosure provides a closed system including a first vial adapted to contain a sample containing a polypeptide, a sample loop adapted to receive the sample from the first vial, a first multiport valve fluidly connected to the sample loop and configured to obtain a quantified amount of the sample through a first port of the first multiport valve, a second multiport valve fluidly connected to the first multiport valve and positioned downstream of the first multiport valve, and a polypeptide binding column configured to be fluidly connected to the second multiport valve when the second multiport valve is in a first position. The polypeptide binding column is configured to bind polypeptides from the sample. The closed system also includes a second vial positioned downstream of the polypeptide binding column, and a buffer source fluidly connected to the first multiport valve and configured to provide an elution buffer solution through the second multiport valve and the polypeptide binding column to the second vial when the second multiport valve is in the first position, such that the elution buffer solution elutes substantially all of the polypeptide from the polypeptide binding column. In some examples, the second vial comprises a flow-through vial configured to filter the elution buffer solution from the elution / polypeptide mixture out of the second vial, thereby leaving only the eluted polypeptide in the second vial.

[0014] Another aspect of the present disclosure provides a closed system including a first vial adapted to contain a sample containing a polypeptide; a sample loop adapted to receive the sample from the first vial; a first multiport valve fluidly connected to the sample loop and configured to obtain a quantified amount of the sample through a first port of the first multiport valve; a second multiport valve fluidly connected to the first multiport valve and positioned downstream of the first multiport valve; and a polypeptide binding column configured to be fluidly connected to the second multiport valve when the second multiport valve is in a first position. The polypeptide binding column is configured to bind polypeptides from the sample. The closed system also includes a second vial positioned downstream of the polypeptide binding column (the second vial comprises a flow-through vial and contains a glycan labeling reagent); and a glycosidase source configured to supply glycosidase (e.g., glucanase) to the polypeptide binding column via the second multiport valve such that the polypeptide bound to the polypeptide binding column enters the glycosidase. The closed system also includes a carrier solution source configured to supply a carrier solution to the polypeptide binding column via a second multiport valve. The carrier solution is configured to release glycans from the polypeptide binding column and transport them to the second vial. The closed system further includes a reaction coil configured to receive a mixture of the glycans released from the second vial via the first multiport valve and a glycan labeling reagent. The reaction coil is configured to incubate the mixture of the released glycans and the glycan labeling reagent to label the glycans. The closed system further includes a third vial positioned downstream of the reaction coil to receive the labeled glycans and the glycan labeling reagent. In some examples, the third vial also includes a flow-through vial configured to substantially filter the glycan labeling reagent from the third vial, thereby leaving substantially only the labeled glycans in the third vial.

[0015] Another aspect of the present disclosure is a method for detecting molecules in a sample vial, comprising: (a) transferring a sample containing molecules from a sample vial to a sample loop; (b) transferring a quantity of the sample from the sample loop to an injection valve; (c) transferring a quantity of the sample from the injection valve to a first multiport valve fluidly coupled to and positioned downstream of the injection valve; (d) transferring a quantity of the sample from the first multiport valve to a trapping column on a second multiport valve when the first multiport valve is in a second position; and (e) capturing molecules in the sample in the trapping column, thereby capturing molecules in the sample. (f) transferring an elution buffer solution from a buffer source to the capture column, thereby eluting the molecules captured by the capture column, and transferring the elution / molecule mixture comprising the elution buffer solution and the eluted molecules to a receiving vial on a third multiport valve positioned downstream of the capture column on the second multiport valve, wherein the receiving vial comprises a flow-through vial; and (g) transferring the molecules in the flow-through vial to an analytical device (column compartment) for analysis of the molecules.

[0016] Another aspect of the present disclosure is a method for detecting polypeptides in a sample, comprising: (a) transferring a sample comprising a polypeptide from a sample vial to a sample loop; (b) transferring a quantity of the sample from the sample loop to an injection valve; (c) transferring a quantity of the sample from the injection valve to a first multiport valve fluidly coupled to and positioned downstream of the injection valve; (d) transferring a quantity of the sample from the first multiport valve to a polypeptide binding column on a second multiport valve when the first multiport valve is in a second position; and (e) capturing polypeptides in the sample in the polypeptide binding column, thereby capturing polypeptides in the sample. (f) transferring an elution buffer solution from a buffer source to the polypeptide binding column, thereby eluting the polypeptide captured by the polypeptide binding column, and transferring the elution / molecular mixture comprising the elution buffer solution and the eluted polypeptide to a receiving vial located downstream of the polypeptide binding column, wherein the receiving vial comprises a flow-through vial; and (g) transferring the polypeptide in the flow-through vial to an analytical device (column compartment) for analysis of the polypeptide.

[0017] Another aspect of the present disclosure provides a method for detecting a polypeptide comprising: (a) transferring a sample comprising a polypeptide from a sample vial to a sample loop; (b) transferring a quantity of the sample from the sample loop to an injection valve; (c) transferring the quantity of the sample from the injection valve to a first multiport valve fluidly coupled to and positioned downstream of the injection valve; (d) transferring the quantity of the sample from the first multiport valve to a polypeptide binding column on a second multiport valve; (e) binding polypeptides in the quantity of the sample to the polypeptide binding column, thereby separating the polypeptides in the sample from the matrix of the sample; (f) transferring a glycosidase (such as a glucanase) through the second multiport valve to the polypeptide binding column to release glycans from the bound polypeptides; and (g) isolating the released polypeptides. The method includes: (h) mixing the released glycans with a glycan labeling reagent in the flow-through vial; (i) transferring the mixture of the released glycans and the glycan labeling reagent through a first multiport valve to a reaction coil on a second multiport valve; (j) incubating the mixture of the released glycans and the glycan labeling reagent in the reaction coil, thereby labeling the glycans; (k) transferring the mixture from the reaction coil to the flow-through vial; and (l) transferring the labeled glycans in the flow-through vial to an analytical device for analysis of the labeled glycans.

[0018] Another aspect of the present disclosure provides a closed system including a sample vial adapted to contain a sample containing a molecule, one or more modifiers, a sample loop adapted to receive the sample from the sample vial, an injection valve fluidly connected to the sample loop and configured to obtain a volume of the sample through its second port, and a first multiport valve fluidly connected to the injection valve and positioned downstream of the injection valve. The closed system includes a capture column configured to be fluidly connected to the second multiport valve when the second multiport valve is in a second position. The capture column is configured to capture molecules from the volume of the sample. The closed system also includes a desalting column configured to be fluidly connected to the second multiport valve when the second multiport valve is in a third position different from the second position. The desalting column is configured to reduce the salt concentration of the molecule. The closed system further includes a reaction coil selectively fluidly connected to the second multiport valve. The reaction coil is configured to receive one or more modifiers and to incubate the molecule and the one or more modifiers to modify the molecule. The receiving vial is positioned to receive a mixture containing the molecules from the capture column, desalting column, or reaction coil, and includes a flow-through vial configured to separate the molecules from the remainder of the mixture so that the molecules can be analyzed.

[0019] Another aspect of the present disclosure is a sample vial adapted to contain a sample comprising a polypeptide, one or more modifiers, A closed system is provided, comprising: a sample loop adapted to receive a sample from a sample vial; an injection valve fluidly connected to the sample loop and configured to obtain a fixed volume of the sample through its second port; a first multiport valve fluidly connected to the injection valve and positioned downstream of the injection valve; and a capture column configured to be fluidly connected to the second multiport valve when the second multiport valve is in a second position. The capture column is configured to capture a polypeptide from the fixed volume of the sample. The closed system also includes a desalting column fluidly connected to the second multiport valve when the second multiport valve is in a third position different from the second position. The desalting column is configured to reduce the salt concentration of the polypeptide. The closed system further includes a reaction coil selectively fluidly connected to the second multiport valve. The reaction coil is configured to receive one or more modifying agents and is configured to incubate the polypeptide and the one or more modifying agents to modify the polypeptide. The closed system further includes a receiving vial downstream of a third multiport valve and fluidly connected to the third multiport valve. The receiving vial is configured to receive the mixture containing the polypeptide from the capture column, desalting column, or reaction coil, and includes a flow-through vial configured to separate the polypeptide from the remainder of the mixture so that the polypeptide can be analyzed.

[0020] Another aspect of the present disclosure provides a closed system including a sample vial adapted to contain a sample comprising a polypeptide, a sample loop adapted to receive the sample from the sample vial, an injection valve fluidly connected to the sample loop and configured to obtain a quantified amount of the sample through its second port, a first multiport valve fluidly connected to the injection valve and positioned downstream of the injection valve, and a polypeptide binding column configured to be fluidly connected to the second multiport valve when the second multiport valve is in its second position. The polypeptide binding column is configured to bind the polypeptide from the sample. The closed system also includes a receiving vial on a third multiport valve positioned downstream of the polypeptide binding column on the second multiport valve, and a buffer source fluidly connected to the first multiport valve and configured to provide an elution buffer solution to the second multiport valve and the polypeptide binding column such that the elution buffer solution elutes substantially all of the polypeptide from the polypeptide binding column into the receiving vial on the third multiport valve when the second multiport valve is in its second position. The receiving vial comprises a flow-through vial configured in combination with a third multi-port valve to bypass the elution buffer solution from the polypeptide binding column / polypeptide mixture, thereby leaving only the eluted polypeptide in the flow-through vial.

[0021] Another aspect of the present disclosure provides a closed system including a sample vial adapted to contain a sample containing a polypeptide; a sample loop adapted to receive the sample from the sample vial; an injection valve fluidly connected to the sample loop and configured to obtain a quantified amount of the sample through its second port; a first multiport valve fluidly connected to the injection valve and positioned downstream of the injection valve; and a polypeptide binding column configured to be fluidly connected to the second multiport valve when the second multiport valve is in a second position. The polypeptide binding column is configured to bind the polypeptide from the sample. The closed system also includes a receiving vial on a third multiport valve positioned downstream of the polypeptide binding column (the receiving vial is a flow-through vial and contains a glycan labeling reagent); and a glycosidase source configured to supply a glycosidase (e.g., glucanase) to the polypeptide binding column through the second multiport valve so that the polypeptide bound to the polypeptide binding column enters the glycosidase. The closed system also includes a carrier solution source configured to supply a carrier solution to the polypeptide binding column through the second multiport valve. The carrier solution releases the glycans from the polypeptide-binding column and transports them to the receiving vial. The closed system further includes a reaction coil configured to receive a mixture of the glycans released from the flow-through vial via the first multiport valve and the glycan labeling reagent. The reaction coil is configured to incubate the mixture of the released glycans and the glycan labeling reagent to label the glycans. The closed system uses the same flow-through vial on a third multiport valve positioned downstream of the reaction coil on the second multiport valve to bypass the glycan labeling reagent and receive the labeled glycans in the flow-through vial. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic diagram of an example of a system for performing online real-time assays constructed in accordance with the teachings of the present disclosure. [Figure 2] FIG. 2 is a schematic diagram of a controller for the system shown in FIG. 1. [Figure 3] FIG. 2 is a schematic diagram illustrating an example of a method for performing a real-time assay of a sample using the system of FIG. 1. [Figure 4] FIG. 2 is a schematic diagram illustrating another example of a method for performing a real-time assay of a sample using the system of FIG. 1. [Figure 5] FIG. 2 is a schematic diagram illustrating another example of a method for performing a real-time assay of a sample using the system of FIG. 1. [Figure 6] FIG. 2 is a schematic diagram illustrating another example of a method for performing a real-time assay of a sample using the system of FIG. 1. [Figure 7] FIG. 2 is a schematic diagram illustrating another example of a method for performing a real-time assay of a sample using the system of FIG. 1. [Figure 8] FIG. 2 is a schematic diagram illustrating another example of a method for performing a real-time assay of a sample using the system of FIG. 1. [Figure 9] FIG. 2 is a schematic diagram illustrating another example of a method for performing a real-time assay of a sample using the system of FIG. 1. [Figure 10] FIG. 1 is a schematic diagram of another example of a system for conducting online real-time assays constructed in accordance with the teachings of the present disclosure. [Figure 11] FIG. 11 is a schematic diagram illustrating an example of a method for performing a real-time assay of a sample using the system of FIG. [Figure 12] FIG. 11 is a schematic diagram illustrating another example of a method for performing a real-time assay of a sample using the system of FIG. [Figure 13] FIG. 11 is a schematic diagram illustrating another example of a method for performing a real-time assay of a sample using the system of FIG. [Figure 14] FIG. 11 is a schematic diagram illustrating another example of a method for performing a real-time assay of a sample using the system of FIG. [Figure 15] FIG. 11 is a schematic diagram illustrating another example of a method for performing a real-time assay of a sample using the system of FIG. [Figure 16] FIG. 11 is a perspective view of an example of a flow-through vial constructed in accordance with the teachings of the present disclosure and usable in the system of FIG. 1 or the system of FIG. 10. [Figure 17] FIG. 17 is a partial cross-sectional view of the flow-through vial of FIG. [Figure 18] 17 is a perspective view of an example of a vial holder constructed in accordance with the teachings of the present disclosure and that can be used in the system of FIG. 1 or the system of FIG. 10 to hold a plurality of flow-through vials of FIG. 16. FIG. [Figure 19] 17 shows a vial holder coupled to the automated sampling system of the system of FIG. 1 or the system of FIG. 10 and receiving one of the flow-through vials of FIG. 16. [Figure 20] 2 is a table showing the results of a study demonstrating the effectiveness of the system of FIG. 1 using three different assays and nine different molecules. [Figure 21] 2 is a graph showing the results of one of three different assays performed with the system of FIG. 1 using one of nine different molecules. [Figure 22] Similar to FIG. 21, but showing the results using the prior art. DETAILED DESCRIPTION OF THE INVENTION

[0023] FIG. 1 shows a schematic diagram of an example of a system 100 constructed in accordance with the teachings of the present disclosure. System 100, which may be located in or within a laboratory (e.g., a testing laboratory) or may be located at a manufacturing facility, is a closed system for automatically or substantially automatically preparing samples of a product containing a molecule of interest and automatically performing assays on the sample, as described in further detail below. By automating (or substantially automating) this process using system 100, assays can be performed in real time (or substantially in real time) and at the manufacturing site (when used at a manufacturing facility), such that the entire process can be performed and the desired results obtained in a few hours (e.g., 2-3 hours), a significant improvement over the five days typically required for previously known, manually operated processes. Furthermore, the closed nature of the process using system 100 maintains sterile conditions. Furthermore, system 100 is a flexible system that can be used to prepare any number of samples (same or different) for various analyses and automatically perform many different assays on those samples, thereby eliminating the need for many different systems to prepare different samples for different analyses. By performing multiple assays within the same system 100, a smaller sample volume may be required than if the same assay were performed on multiple systems, which generally results in some sample loss as dead volume. Thus, it is contemplated that the systems 100 described herein can conserve sample by minimizing or avoiding dead volume. Furthermore, the systems 100 described herein can perform multiple assays while maintaining a smaller footprint than individual systems configured to perform each assay individually. For example, the systems described herein with a capture column (e.g., a Protein A column) within valve 112 can achieve the functionality of large-scale purification while using a smaller footprint.

[0024] The system 100 shown in FIG. 1 generally includes one or more sample vials, needles, sample loops, a metering device, a needle seat port, a first column, a second column, one or more buffer sources, one or more variegated reagent sources, one or more carrier solution sources, a first reaction coil (coil A in FIG. 1 ), a second reaction coil (coil B in FIG. 1 ), and one or more receiving vials, as well as four multiport valves 104, 108, 112, and 116 and conduits (e.g., stainless steel conduits) extending between the components of the system 100, as needed, to facilitate fluid communication between the components of the system 100. The system 100 shown in FIG. 1 also includes an analytical device, a first pump 120, and a second pump 124. Nevertheless, it will be understood that the system 100 may not include one or more of the above-listed components and / or additional components. The second reaction coil (e.g., coil B in FIG. 1 ) may optionally be omitted from the system 100.

[0025] Each of the one or more sample vials is adapted to contain a sample containing a molecule of interest. Preferably, the sample is obtained (e.g., delivered) automatically using an automated sampling system (e.g., the autosampler used in the Agilent 1290 Infinity II product), although the sample can also be obtained manually. In some examples, the molecule is a polypeptide or polynucleotide, such as a therapeutic polypeptide (described in more detail below). In other examples, the molecule is a small molecule (i.e., having a molecular weight of about 900 daltons or less and capable of diffusing across a cell membrane within a reasonable time). A small molecule can, for example, comprise, consist essentially of, or consist of a metabolite. Each of the one or more sample vials is preferably contained within the automated sampling system itself and generally takes the form of a flow-through vial, the details of which are described in more detail herein, although the sample vial can also be other vials (e.g., a non-flow-through vial). In any case, if desired, a needle is movable to automatically withdraw a sample from one of the sample vials and place the sample into a sample loop. The sample loop acts as a temporary reservoir or buffer zone for the applied sample, while at the same time preventing the sample from entering the metering device, which in this example takes the form of an analytical head, arranged to control the amount of sample drawn by the needle and applied to the sample loop, which is finally forced from the sample loop into the needle seat port.

[0026] In this example, multiport valve 104 takes the form of a six-port injection valve. Multiport valve 104 is movable into fluid communication with a needle seat port as needed. The sample then flows from the needle seat port through multiport valve 104 to another downstream component of system 100 (the specific component will depend on the assay being performed). In some cases, the sample flows from the needle seat port through multiport valve 104 to an analytical device. In some cases, the sample flows from the needle seat port through multiport valve 104 to multiport valve 108.

[0027] In this example, multiport valve 108 takes the form of a six-port injection valve that is in selective fluid communication with multiport valve 104 such that sample (and / or buffer from a buffer solution source or variance reagent from a variance reagent source) can flow from the needle seat ports through multiport valve 104 and into multiport valve 108.

[0028] The multiport valve 112 in this example is a four-position ten-port valve, the position of which determines whether the multiport valve 108 is fluidly coupled to the first column or the second column (both downstream of the multiport valve 108). When the multiport valve 112 is in the first position, the first column is fluidly coupled to the multiport valve 108 (and in turn to the multiport valve 104), and the second column is fluidly isolated from the multiport valve 108, such that the first column may receive a sample, a buffer, a variator reagent, a carrier solution, or a combination thereof. Conversely, when the multiport valve 112 is in a second position different from the first position, the second column is fluidly coupled to the multiport valve 108 (and in turn to the multiport valve 104), and the first column is fluidly isolated from the multiport valve 108, such that the second column may receive a sample, a buffer, a variator reagent, and / or a carrier solution. It is contemplated that the multiport valve 112 of the systems and methods described herein can be configured to perform analytical assays, such as column-based assays. In the systems and methods described herein, the first column can include or consist of an analytical column. For example, the analytical column can be a column of a chromatography device as described herein, or an incubation column. The incubation column can be configured for protein digestion according to a MAM as described herein.

[0029] The first column in this example is a capture column configured to capture molecules of interest from the sample, thereby substantially separating the molecules in the sample from the sample matrix (which can then be discarded). In some cases, the capture column can also serve to preconcentrate the molecules (e.g., by clustering the molecules in the capture column), although this step is not required. When the molecule is a polypeptide, the capture column takes the form of a polypeptide-binding column selected from the group consisting of a Protein A column, a Protein G column, a Protein A / G column, a Protein L column, an amino acid column, an avidin column, a streptavidin column, a carbohydrate-binding column, a carbohydrate column, a glutathione column, a heparin column, a hydrophobic interaction column, an immunoaffinity column, a nucleotide / coenzyme column, a specialty column, and an immobilized metal affinity chromatography (IMAC) column. For example, a Protein A column is useful for polypeptides that are human IgG, IgM, IgA, or IgE subclasses 1, 2, or 4 (and that include the human Fc portion and / or Fab region of the human VH3 family). Protein G can be used to purify human IgG subclasses 1-4. Recombinant fusion protein A / G can also be used to purify all of these human antibody classes because the fusion protein provides binding sites for both Protein A and Protein G. Thus, Protein A / G fusion proteins can be used to purify human IgG, IgA, IgE, and IgM. Furthermore, Protein L can be used to purify human IgG, IgM, IgA, IgE, and IgD, provided that the target antibody has the appropriate kappa (κ) subtype light chain (i.e., VκI, VκIII, and VκIV subtypes). Protein L also binds to the variable (V) chain of the antibody, and therefore can be used to purify Fab and scFv fragments that also have the appropriate κ chain subtype. However, if the molecule is a small molecule, the capture column can instead take the form of a reverse-phase column, size-exclusion column, ion-exchange column, normal-phase column, chiral separation column, mixed-mode column, or hydrophobic interaction column.For example, if the molecule is a small molecule such as a metabolite, the capture column can take the form of a trimodal (or three-phase) chromatography column, including reversed-phase, cationic (cation exchange), and anionic (anion exchange). Analytes can be eluted by pH, ionic strength, and / or organic strength gradients. Elution buffers for analysis of small molecules (such as metabolites) can include aqueous, low-salt, volatile buffers. In some instances, the low-salt, volatile buffers include organic solvents. The organic solvent levels can be lower than those used in HILIC. By way of example, typical starting solvents for HILIC can have an organic concentration of ≥ 50% (v / v).

[0030] Meanwhile, the second column in this example is a desalting column equilibrated with a buffer (e.g., a proteolysis buffer) so as to reduce the salt concentration of the molecule. Thus, for example, if the molecule is a polypeptide, the desalting column is configured to reduce the salt concentration of the polypeptide (i.e., desalt the polypeptide). The second column preferably takes the form of a size exclusion chromatography column, although other chromatography columns can be used instead.

[0031] The needle is also movable to obtain buffer solution from one or more buffer sources and to deposit the buffer solution into the sample loop. The buffer solution can then flow through multiport valve 104 and into multiport valve 108 in a manner similar to that described above (with respect to sample flow). The buffer solution can then be directed to appropriate components of system 100. In one example, the buffer solution can be an elution buffer solution (e.g., a volatile buffer (e.g., a low-salt volatile buffer such as water and formic acid; water and acetonitrile and ammonium acetate; or water and acetonitrile and ammonium bicarbonate), an acidic buffer in the case of a protein A-bound antibody, or a strongly acidic (pH 3 or less) buffer in the case of a protein G-bound antibody), which is ultimately delivered to the first column when multiport valve 112 is in the first position. As the elution buffer solution flows into and through the first column, it elutes substantially all of the molecules (e.g., polypeptides) captured on the first column.

[0032] The needle is also movable to obtain change reagent from one or more change reagent sources and place the change reagent into the sample loop. The change reagent can then flow through multiport valve 104 into multiport valve 108 in a manner similar to that described above (with respect to sample flow). The change reagent can then be directed to the appropriate components of system 100 to change the molecule in the desired manner.

[0033] Generally speaking, the altering reagent is selected from the group consisting of a denaturing reagent, a reducing reagent, an alkylating reagent, a solution containing (e.g., consisting of) a glycosidase (e.g., glucanase), a glycan labeling reagent, a quenching reagent, an enzyme, and combinations thereof.

[0034] Denaturing reagents can be used to denature molecules (e.g., polypeptides) in a sample. In examples where the denaturing reagent is or includes a denaturing reagent, the denaturing reagent can be or include a denaturing detergent or chaotrope. In examples where the denaturing reagent is or includes a denaturing detergent, the denaturing detergent is preferably selected from the group consisting of sodium dodecyl sulfate (SDS), sodium cholate, sodium deoxycholate, sodium glycocholate, sodium taurocholate, sodium taurodeoxycholate, N-lauroylsarcosine, lithium dodecyl sulfate, hexadecyltrimethylammonium bromide (CTAB), and trimethyl(tetradecyl)ammonium bromide (TTAB). More preferably, the denaturing detergent is SDS. In variations where the denaturing reagent is or includes a chaotrope, the chaotrope is preferably selected from the group consisting of urea, n-butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, and thiourea. Alternatively or additionally, the denaturing reagent may be or include a heated fluid having a temperature suitable to reach, if not maintain, a predetermined temperature (e.g., about 22°C to about 120°C).

[0035] A reducing reagent can be utilized to cleave disulfide bond bridges, thereby reducing molecules (e.g., polypeptides). The reducing reagent can be selected from the group consisting of dithiothreitol (DTT), glutathione, β-mercaptoethanol (β-ME), and tris(2-carboxyethyl)phosphine (TCEP). Although not shown here, the reducing reagent can be provided by a cooled container (e.g., a refrigerator having a temperature of 4°C). Meanwhile, when utilized, an alkylating agent alkylates sulfhydryls, thereby alkylating molecules (e.g., polypeptides). The alkylating agent is preferably an alkylating agent such as indole-3-acetic acid (IAA), although other alkylating agents can also be used.

[0036] A solution containing glycosidase can be utilized to induce glycosidase on the molecule (e.g., polypeptide) captured by the first column and inject the glycosidase, which then releases glycans in the captured molecule (e.g., polypeptide) from the captured molecule (e.g., polypeptide) into the solution. The glycosidase in the solution is preferably selected from the group consisting of glucanase, endoglycosidase, glycosamidase, and O-glycanase, and combinations thereof (e.g., endoglycosidase, glycosamidase, and O-glycanase, and combinations thereof). When the glycosidase is or includes an endoglycosidase, the endoglycosidase can be selected from the group consisting of endoglycosidase D, endoglycosidase F (endoglycosidase F1, endoglycosidase F2, and endoglycosidase F3, and combinations thereof), endoglycosidase H, endoglycosidase B, endoglycosidase C, endoglycosidase D, endoglycosidase F (endoglycosidase F1, endoglycosidase F2, and endoglycosidase F3, and combinations thereof), endoglycosidase H, endoglycosidase F ... Preferably, the glycosidase is selected from the group consisting of endoglycosidase S, endoglycosidase M, and endoglycosidase B. When the glycosidase is or comprises a glycosamidase, the glycosamidase is preferably selected from the group consisting of glycopeptidase, peptide N-glycosidase, PNGase, N-glycohydrolase, and N-glycanase. When the glucanase is or comprises PNGase, the PNGase preferably comprises peptide:N-glycosidase F (PNGF). When the glycosidase is or comprises an O-glycanase, the O-glycanase is preferably endoGalNAc-ase D or endoGalNAc-ase A.

[0037] As the glycans are released into solution in this manner, the needle can be further moved to obtain a carrier solution (e.g., deionized (DI) water) from one or more carrier solution sources and place the carrier solution into the sample loop. The carrier solution can then flow through multiport valve 104 and into multiport valve 108 in a manner similar to that described above (with respect to sample flow). The carrier solution can then be fed, for example, to a first column, passing through the first column and carrying the released glycans from the first column.

[0038] The glycans released from the molecule (e.g., polypeptide) and carried through the first column can then be labeled (e.g., fluorescently labeled) using a glycan-labeling reagent, preferably in the form of a fluorophore (e.g., selected from the group consisting of 2-aminobenzoic acid, 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt, 8-aminonaphthalene-1,3,6-trisulfonic acid trisodium salt, anthranilamide, and 4-methoxybenzamidine) or a chromophore (e.g., 3-methyl-1-phenyl-2-pyrazolin-5-one or phenylhydrazine).

[0039] Enzymes can be used to digest molecules (e.g., polypeptides). The enzymes preferably take the form of proteases, cellulases, lipases, amylases, glucoamylases, glucose isomerases, xylanases, phytases, arabinanases, polygalacturonanases, hydrolases, chymosin, ureases, pectinases, or β-glucanases. Alternatively, quenching reagents can be used to terminate reactions (e.g., enzymatic reactions) by altering the pH conditions of the sample. The quenching reagent preferably takes the form of trifluoroacetic acid (TFA), although other quenching reagents can be used instead.

[0040] Multiport valve 116 in this example is a four-position ten-port valve, the position of which determines whether multiport valve 108 is fluidly coupled to a first reaction coil or a second reaction coil (both downstream of multiport valve 108). When multiport valve 116 is in the first position, the first reaction coil is fluidly coupled to multiport valve 108 (and multiport valve 104), and the second reaction coil is fluidly isolated from multiport valve 108 such that the first reaction coil can receive a sample or a portion thereof (e.g., glycans released from polypeptides in the sample), a buffer, a denaturing reagent, a carrier solution, or a combination thereof. Conversely, when multiport valve 116 is in a second position different from the first position, the second reaction coil is fluidly connected to multiport valve 108 (and in turn multiport valve 104), and the first reaction coil is fluidly isolated from multiport valve 108 so that the second reaction coil can receive a sample or a portion thereof, a buffer, and / or a modified reagent.

[0041] Each of the first and second reaction coils is configured to incubate a molecule (e.g., a polypeptide) and one or more modifying agents to modify the molecule (e.g., the polypeptide). The first or second reaction coil can, for example, receive a quenching reagent and the polypeptide and incubate the quenching reagent and the polypeptide to facilitate quenching of the polypeptide. As another example, the first or second reaction coil can receive a denaturing reagent and the polypeptide and incubate the denaturing reagent and the polypeptide to facilitate denaturation of the polypeptide. As another example, the first or second reaction coil can receive a reducing reagent and the polypeptide and incubate the reducing reagent and the polypeptide to facilitate reduction of the polypeptide. As yet another example, the first or second reaction coil can receive an alkylating reagent and the polypeptide and incubate the alkylating reagent and the polypeptide to facilitate alkylation of the polypeptide. As a further example, the first or second reaction coil can receive a mixture containing released glycans and a glycan labeling reagent and incubate the mixture to facilitate labeling (e.g., fluorescent labeling) of the released glycans.

[0042] To facilitate the desired incubation, system 100 may further include a heating element positioned immediately adjacent to or otherwise thermally coupled to the first and / or second reaction coils. The heating element may take the form of, for example, a heating coil, an induction heater, a heat pump, a cartridge heater, electrical resistance wire, or other element suitable for heating one or more portions of each of the first and second reaction coils. Thus, when system 100 includes a heating element thermally coupled to the first reaction coil, the heating element is configured to maintain the first reaction coil at a first predetermined incubation temperature, which may be, for example, about 30°C, about 40°C, about 80°C, or some other incubation temperature, depending on the application. Similarly, when system 100 includes a heating element thermally coupled to the second reaction coil, the heating element is configured to maintain the second reaction coil at a second predetermined incubation temperature, which may be the same as or different from the first predetermined incubation temperature.

[0043] The one or more receiving vials in this example are generally positioned downstream of all other components of system 100. Like the one or more sample vials, the one or more receiving vials generally take the form of one or more flow-through vials, although other vials (e.g., non-flow-through vials) can be used as well. The one or more receiving vials are generally fluidly coupled to multiport valve 112 and / or multiport valve 116. Thus, the one or more receiving vials are generally positioned to receive a mixture containing molecules (e.g., polypeptides) from one or more of the first column, the second column, the first reaction coil, and the second reaction coil. In the illustrated example, system 100 utilizes two flow-through vials: a first vial fluidly coupled to multiport valve 112 and a second vial fluidly coupled to multiport valve 116. Thus, the first flow-through vial is positioned to receive a mixture containing molecules from either the first column or the second column (depending on which column is being used). The mixture can include, for example, an elution buffer solution from the first column and molecules eluted by the elution buffer solution, while the second flow-through vial is positioned to receive a mixture containing molecules from either the first reaction coil or the second reaction coil (depending on which coil is used). The mixture can include, for example, molecules denatured and reduced in the first reaction coil.

[0044] It will be appreciated that in some examples, each of the receiving vials may have a plurality of holes positioned and sized to substantially prevent the passage of molecules in the received mixture while allowing the remainder of the mixture to pass and exit each receiving vial. Thus, the holes in each of the receiving vials effectively separate the molecules in the receiving mixture from the remainder of the mixture. The separated molecules can then be sent to an analytical device (to analyze the molecules) or to other components in system 100 for further preparation, while the remainder of the mixture can then be discarded. Alternatively or additionally, this separation function may be performed by timely movement of one or more of multiport valve 104, multiport valve 108, multiport valve 112, and multiport valve 116.

[0045] 1, system 100 can also include one or more intermediate vials, each containing a fluid configured to dilute the salt concentration in the sample. The fluid can take the form of, for example, water, a buffer (e.g., a low-salt buffer), an organic solvent (e.g., methanol, ethanol, propanol, acetone), or a mobile phase that acts as a diluent. In either case, when it is necessary to dilute the salt concentration in the sample, the needle can be moved to either (i) obtain a sample from an appropriate component in system 100 and deposit the sample in each intermediate vial for dilution, or (ii) obtain a fluid from each intermediate vial and deposit the fluid in the component of system 100 containing the sample.

[0046] The first pump 120 in this example takes the form of a binary or quaternary pump fluidly coupled to the multiport valve 104. The binary or quaternary pump is generally positioned to assist in moving material through the multiport valve 104 and to other components of the system 100. In this example, the pump is positioned to drive sample from the needle seat port and drive mobile phase to the first column, second column, or other components of the system 100, as needed.

[0047] The second pump 124 in this example takes the form of a quaternary pump fluidly coupled to the multiport valve 108. The second pump 124 is generally positioned to help clean various components of the system 100. In this example, the second pump 124 is positioned to drive various solutions (e.g., buffers) through the first and second columns to wash and equilibrate the columns, and to drive various solutions through the first and second reaction coils to wash the coils and prevent carryover from sample to sample. For example, the second pump 124 may be configured to wash the flow-through vials in-situ, so that the flow-through vials do not need to be removed for washing, thus simplifying the design of the system 100 and minimizing the number of moving parts within the system 100.

[0048] The analytical device is generally configured to analyze molecules (e.g., polypeptides) after they have been prepared by system 100. The analytical device can take the form of, for example, a liquid chromatography device (e.g., an ion exchange chromatography column, a cation exchange chromatography column, an anion exchange chromatography column), a high performance liquid chromatography device, an ultra-high performance liquid chromatography device, a mass spectrometry device (e.g., a high resolution accurate mass (HRAM) mass spectrometer), a spectrophotometric device (e.g., a UV detector), a glycan analysis device, another type of analytical device, or a combination thereof. It is contemplated that an HRAM mass spectrometer may be useful for analyzing small molecules, such as metabolites, as described herein. It will also be understood that many analytical devices may be utilized in system 100.

[0049] 1, system 100 also includes a controller, which in this example is communicatively coupled or connected to the various components of system 100 and monitors and facilitates or directs the above-described operation of system 100 by sending signals (e.g., control signals, data) to and receiving signals (e.g., data) from the various components of system 100. The controller may be located immediately adjacent to the other components of system 100 (e.g., in the same environment as system 100) or may be located remotely from the other components of system 100.

[0050] As used herein, the phrases “communicatively coupled” and “connected” are defined to mean directly coupled or connected, or indirectly coupled or connected through one or more intermediate components. Such intermediate components may include hardware and / or software-based components. It is understood that the controller may be communicatively coupled or connected to various components of system 100 via one or more wireless networks, one or more wired networks, a combination of one or more wired and wireless networks (e.g., cellular networks and / or 802.11x-compliant networks), and may include publicly accessible networks such as the Internet, private networks, or a combination thereof. The type and configuration of the networks are implementation dependent, and any type of communication network now available or later developed that facilitates the described communications between the controller and the components of system 100 may be used.

[0051] As shown in FIG. 2 , the controller includes a processor 352, a memory 356, a communications interface 360, and computational logic 364. The processor 352 may be a general-purpose processor, a digital signal processor, an ASIC, a field-programmable gate array, a graphics processing unit, an analog circuit, a digital circuit, or any other known or later-developed processor. The processor 352 operates according to instructions in the memory 356. The memory 356 may be volatile or nonvolatile memory. The memory 356 may include one or more of read-only memory (ROM), random-access memory (RAM), flash memory, electronically erasable programmable read-only memory (EEPROM), or other types of memory. The memory 356 may include optical, magnetic (hard drive), or any other form of data storage device.

[0052] A communications interface 360 ​​is provided to enable or facilitate electronic communications between the controller and components of system 100 over one or more utilization networks. Communications interface 360 ​​may be or include, for example, one or more Universal Serial Bus (USB) ports, one or more Ethernet ports, and / or one or more other ports or interfaces. Electronic communications may occur via any known communications protocol, including, by way of example, USB, RS-232, RS-485, WiFi, Bluetooth, and / or any other suitable communications protocol.

[0053] Logic 364 generally includes one or more control routines and / or one or more subroutines embodied as computer-readable instructions stored in memory 356. The control routines and / or subroutines may implement PID (proportional-integral-derivative), fuzzy logic, non-linear, or any other suitable type of control. Processor 352 generally executes logic 364 and performs actions related to the operation of system 100.

[0054] Generally, logic 364, when executed, causes processor 352 to control the components of system 100, particularly multiport valves 104, 108, 112, 116, needles, pumps 120, 124, and heating elements, such that system 100 operates in the desired manner described herein. As an example, logic 364, when executed, can cause processor 352 to move multiport valve 112 and / or multiport valve 116 to or between any of the positions described herein, thereby fluidly coupling the various components of system 100, as described above.

[0055] In other variations, logic 364 may cause additional, fewer, and / or different functions to be performed when executed by processor 352. Furthermore, in other variations, logic 364 may be executed by processor 352 in an order different from that described herein. Finally, because system 100 may be used to perform real-time analysis of multiple samples (from the same product and / or different products), it is understood that logic 364 may be executed by processor 352 any different number of times.

[0056] 3 illustrates an example method 300 for automatically (or substantially automatically) performing a first real-time assay of a sample using system 100. In this example, the assay is a production titer, and method 300 generally includes: (1) moving (e.g., automatically) a needle to obtain a sample from one of the sample vials and place the sample in a sample loop, (2) forcing the sample from the sample loop into a needle seat port, (3) moving the sample from the needle seat port through multiport valve 104 to an analytical device (in this example, taking the form of a spectrophotometric detector), and (4) determining the titer / concentration of the sample using the analytical device.

[0057] 4 illustrates another example method 400 for automatically (or substantially automatically) performing a second real-time assay on a sample using system 100. In this example, the assay is an assessment of agglutination, and method 400 generally includes: (1) moving (e.g., automatically) a needle to obtain a sample from a first vial (one of the sample vials) and place the sample in a sample loop; (2) forcing the sample from the sample loop into a needle seat port; (3) moving the sample from the needle seat port through multiport valve 104 to multiport valve 108; (4) moving the sample from multiport valve 108 to a first column through multiport valve 112 (in a first position); and (5) capturing molecules in the sample in the first column, thereby separating the molecules in the sample from the sample matrix, which flows out of the first column and is fluidly connected to the first column. (6) transferring an elution buffer solution from the buffer source to the first column via the needle, needle seat port, and multiport valves 104, 108, and 112, thereby eluting the molecules captured by the first column; (7) transferring an elution / molecule mixture comprising the elution buffer solution and the eluted molecules to a second vial (one of the receiving vials), which separates the molecules in the mixture from the remainder of the mixture; (8) transferring the molecules from the second vial to an analytical device via the needle, needle seat port, and multiport valve 104; and (9) quantifying the level of aggregation in the molecules using the analytical device.

[0058] FIG. 5 illustrates another example method 500 for automatically (or substantially automatically) performing a third real-time assay on a sample using system 100.In this example, the assay is a charge mutation profile, and method 500 generally includes: (1) moving (e.g., automatically moving) a needle to obtain a sample from a first vial (one of the sample vials) and place the sample in a sample loop; (2) forcing the sample from the sample loop into the needle seat port; (3) moving the sample from the needle seat port through multiport valve 104 to multiport valve 108; (4) moving the sample from multiport valve 108 to a first column through multiport valve 112 (in a first position); and (5) discharging the sample from the first column. (5) capturing molecules in the sample in the first column, thereby separating the molecules in the sample from the sample matrix, which flows out of the first column and is discarded via a second vial (one of the receiving vials) fluidly connected to the first column; (6) transferring an elution buffer solution from a buffer source to the first column via the needle, needle seat port, and multiport valves 104, 108, and 112, thereby eluting the molecules captured by the first column; and (7) discharging an elution / molecule mixture comprising the elution buffer solution and the eluted molecules into the second vial. (7) transferring the molecules from the second vial to a third vial (one of the intermediate vials) to separate the molecules in the mixture from the remainder of the mixture, thereby producing an affinity-purified sample in the second vial; (8) transferring the molecules from the second vial to a third vial (one of the intermediate vials) to dilute the affinity-purified sample; (9) transferring the affinity-purified sample from the third vial to multiport valve 108 via the needle, needle seat port, and multiport valve 104; and (10) transferring the affinity-purified sample from multiport valve 108 to a second column via multiport valve 104. (11) applying the affinity purified sample to a second column, thereby further reducing the salt concentration of the sample; (12) transferring this further diluted affinity purified sample from the second column back to the second vial; (13) transferring the affinity purified sample from the second vial to an analytical device via the needle, needle seat port, and multiport valve 104; and (14) performing charge variation assessment using the analytical device (which in this example takes the form of an ion exchange column).

[0059] FIG. 6 illustrates another example method 600 for automatically (or substantially automatically) performing a fourth real-time assay on a sample using system 100.In this example, the assay is a glycosylation profiling assay, and method 600 generally includes: (1) moving (e.g., automatically moving) a needle to obtain a sample from a first vial (one of the sample vials) and place the sample into a sample loop; (2) forcing the sample from the sample loop into the needle seat port; (3) moving the sample from the needle seat port through multiport valve 104 to multiport valve 108; and (4) moving the sample from multiport valve 108 to a first column through multiport valve 112 (in a first position). (5) capturing molecules in the sample in the first column, thereby separating the molecules in the sample from the sample matrix, which flows out of the first column and is discarded via a second vial (one of the receiving vials) fluidically connected to the first column; and (6) transferring a solution containing glycosidase (in this example, PNGase-F) from the source to the first column via the needle, needle seat port, and multiport valves 104, 108, and 112, thereby releasing glycans from the molecules captured by the first column. (7) transferring the released glycans to a second vial (one of the receiving vials); (8) transferring a glycan labeling reagent (in this example, 2-AA) from the source to the second vial via the needle; (9) mixing the glycan labeling reagent and the released glycans in the second vial; (10) transferring the mixture of the released glycans and glycan labeling reagent to a second reaction coil (coil B) via the needle, multiport valves 104, 108, and multiport valve 112 (in the fourth position); (11 (12) transferring the mixture from the second reaction coil to a third vial (another one of the receiving vials), which separates the labeled glycans in the mixture from the remainder of the mixture; (13) transferring the labeled glycans from the third vial to an analytical device via the needle, needle seat port, and multiport valve 104; and (14) performing normal phase chromatographic separation and quantification using the analytical device.

[0060] 7 shows another example method 700 for automatically (or substantially automatically) performing a fifth real-time assay on a sample using system 100. In this example, the assay is a MAM assay, and method 700 generally includes (1) moving (e.g., automatically) a needle to obtain a sample from a first vial (one of the sample vials) and place the sample into a sample loop, (2) forcing the sample from the sample loop into the needle seat port, (3) moving the sample from the needle seat port through multiport valve 104 to multiport valve 108, and (4) discharging the sample from multiport valve 108 to a first column through multiport valve 112 (in a first position). (5) capturing molecules in the sample in the first column, thereby separating the molecules in the sample from the sample matrix, which flows out of the first column and is discarded through a second vial (one of the receiving vials) fluidly connected to the first column; and (6) transferring an elution buffer solution from the buffer source to the first column through the needle, needle seat port, and multiport valves 104, 108, and 112, thereby eluting the molecules captured by the first column. (7) transferring the elution / molecule mixture, including the elution buffer solution and the eluted molecules, to a second vial (one of the receiving vials), which separates the molecules in the mixture from the remainder of the mixture, thereby generating an affinity-purified sample in the second vial; (8) transferring the affinity-purified sample from the second vial to a third vial (one of the intermediate vials) via a needle to dilute the affinity-purified sample; and (9) transferring the diluted affinity-purified sample from the third vial back to the second vial via a needle. (10) transferring the denaturing reagent and reducing reagent to a second vial via the needle; (11) transferring the affinity purified sample, denaturing reagent, and reducing reagent from the second vial to a first reaction coil (coil A) via the needle, multiport valves 104, 108, and multiport valve 112 (in a third position); (12) incubating the mixture in the first reaction coil, thereby denaturing and reducing the molecules; and (13) transferring the denatured and reduced molecules from the first reaction coil.(14) transferring the alkylating reagent to the fourth vial (another one of the receiving vials) fluidly connected to the first reaction coil; (15) transferring the modified and reduced molecule and the alkylating reagent from the fourth vial to the first reaction coil (coil A) via the needle, multiport valves 104, 108, and multiport valve 112 (which are in the third position); (16) incubating the modified and reduced molecule and the alkylating reagent in the first reaction coil, thereby alkylating the modified and reduced molecule; (17) transferring the modified, reduced, and alkylated molecule from the first reaction coil to the fourth vial; and (18) transferring the modified, reduced, and alkylated molecule from the fourth vial to the second column via the needle, needle seat port, multiport valves 104, 108, and multiport valve 112 (which are moved from the first position to the second position). (19) applying the modified, reduced, and alkylated molecules to a second column, thereby reducing the salt concentration of the molecules; (20) returning the desalted molecules from the second column to a second vial; (21) (either before step (20) or after step (20)) transferring the enzyme to the second vial via the needle; and (22) transferring the desalted molecules and enzyme from the second vial to a second reaction coil (coil B) via the needle, multiport valves 104, 108, and multiport valves. (23) incubating the desalted molecules and enzyme in a second reaction coil, thereby digesting the molecules; (24) returning the digested molecules from the second reaction coil to the fourth vial; (25) transferring the digested molecules from the fourth vial to an analytical device via the needle, needle seat port, and multiport valve 104; and (26) analyzing the digested molecules using the analytical device.

[0061] FIG. 8 illustrates, in part, another example method 800 for automatically (or substantially automatically) performing a sixth real-time assay on a sample using system 100.In this example, the assay is a synthesis product assay, and method 800 generally includes: (1) moving (e.g., automatically moving) a needle to obtain a sample from a first vial (one of the sample vials) and place the sample into a sample loop; (2) forcing the sample from the sample loop into the needle seat port; (3) moving the sample from the needle seat port through multiport valve 104 to multiport valve 108; and (4) moving the sample from multiport valve 108 to a first column through multiport valve 112 (which is in a first position). (5) capturing molecules in the sample in the first column, thereby separating the molecules in the sample from the sample matrix, which flows out of the first column and is discarded via a second vial (one of the receiving vials) fluidically connected to the first column; (6) transferring an elution buffer solution from a buffer source to the first column via the needle, needle seat port, and multiport valves 104, 108, and 112, thereby eluting the molecules captured by the first column; and (7) transferring the elution buffer solution from the buffer source to the first column via the needle, needle seat port, and multiport valves 104, 108, and 112, thereby eluting the molecules captured by the first column. (8) transferring the elution / molecule mixture containing the solution and the eluted molecules to a second vial (one of the receiving vials), which separates the molecules in the mixture from the remainder of the mixture, thereby producing an affinity-purified sample in the second vial; (9) transferring the reaction stop reagent to the second vial via the needle; and (10) transferring the affinity-purified sample and reaction stop reagent from the second vial to a first reaction coil (coil A) via the needle, multiport valves 104, 108, and multiport valve 112 (in a third position). (10) incubating the affinity purified sample and quenching reagent in the first reaction coil, thereby quenching the reaction of the molecules; (11) transferring the quenched molecules from the first reaction coil to a third vial (another one of the receiving vials) fluidically connected to the first reaction coil; (12) transferring the quenched molecules from the third vial to an analytical device via the needle, needle seat port, and multiport valve 104; and (13) analyzing the quenched molecules using the analytical device.

[0062] 9 illustrates another example method 900 for automatically (or substantially automatically) performing a seventh real-time assay on a sample using system 100. In this example, the assay is a metabolite analysis, and method 900 generally includes: (1) moving (e.g., automatically moving) a needle to obtain (e.g., automatically obtain) a sample from a first vial (one of the sample vials) and place the sample into a sample loop; (2) forcing the sample from the sample loop into a needle seat port; (3) moving the sample from the needle seat port through multiport valve 104 to multiport valve 108; (4) moving the sample from multiport valve 108 to a first column (in this example, taking the form of a three-mode chromatography column) through multiport valve 112 (in a first position); and (5) capturing molecules in the sample in the first column, thereby separating the molecules in the sample from the sample matrix, which flows out of the first column and into a second vial (one of the receiving vials) fluidically connected to the first column. (6) transferring both an elution buffer solution (e.g., a low-salt volatile buffer solution) from the buffer source and an organic solvent from one of the intermediate vials to the first column via the needle, needle seat port, and multiport valves 104, 108, and 112, thereby eluting and diluting the molecules captured by the first column; (7) transferring an elution / molecule mixture comprising the elution buffer solution, the organic solvent, and the eluted molecules to a second vial (one of the receiving vials), which separates the molecules in the mixture from the remainder of the mixture; (8) transferring the molecules from the second vial to an analytical device (in this example, taking the form of a mass spectrometry device such as an HRAM mass spectrometer) via the needle, needle seat port, and multiport valve 104; and (9) analyzing the molecules for metabolites using the analytical device. It will be appreciated that because the first column in this example is a trimodal chromatography column, method 900 advantageously facilitates the analysis of metabolites without the use of differential labels or ion-pairing reagents.It will also be appreciated that method 900 can be used to analyze over 200 different metabolites, including, for example, amino acids (and highly hydrophilic amino acids such as leucine isomers), vitamins, nucleotide-sugars, nucleoside analogs, keto acids, carbohydrates, amino alcohols, nucleotides, polyamines, and phospholipids.

[0063] 10 shows a schematic diagram of another example system 1000 assembled in accordance with the teachings of the present disclosure. The system 1000 shown in FIG. 10 is similar to the system 100 in that the system 1000 also generally includes one or more sample vials, needles, sample loops, a metering device, a needle seat port, a first column, a second column, one or more buffer sources, one or more sensitizing reagent sources, one or more carrier solution sources, a reaction coil, a heating element thermally coupled to the reaction coil, one or more receiving vials (e.g., one or more flow-through vials), as well as four multiport valves 104, 108, 112, 116, a controller, an analytical device, a first pump 120, and a second pump 124 used in the system 100 of FIG. 1, and, if necessary, conduits (e.g., stainless steel conduits) extending between the components of the system 1000 to facilitate fluid communication between the components of the system 1000. However, system 1000 shown in FIG. 10 differs from system 100 shown in FIG. 1 in two main respects. First, unlike system 100, system 1000 does not include a second reaction coil. Second, some of the components described above are arranged differently in system 1000 than in system 100. As one example, in system 1000, the analyzer, first pump 120, and second pump 124 are fluidly coupled to ports of multiport valve 108 instead of ports of multiport valve 104 (as in system 100). As another example, in system 1000, the reaction coil (and a heating element thermally coupled to the reaction coil) are fluidly coupled to ports of (and between) multiport valve 112 instead of ports of multiport valve 116 (as in system 100). Thus, in system 1000, it is the position of multiport valve 112 (and not the position of multiport valve 116) that determines whether multiport valve 108 is fluidly coupled to the reaction coil (and heating element).

[0064] Despite these differences, system 1000, like system 100, is a closed system for automatically or substantially automatically preparing samples of molecule-containing products for analysis and automatically performing assays on those samples. By automating (or substantially automating) this process using system 1000, the assays can be performed in real time (or substantially in real time) and at the manufacturing site (when used in a manufacturing facility), such that the entire process can be performed and the desired results obtained in a few hours (e.g., 2-3 hours), a significant improvement over the days to weeks typically required for previously known manually operated processes. Furthermore, the closed nature of the process using system 1000 maintains sterile conditions. Furthermore, like system 100, system 1000 is a flexible system that can be used to prepare any number of samples (same or different) for various analyses and automatically perform many different assays on those samples, thereby eliminating the need for many different systems to prepare different samples for different analyses.

[0065] 11 shows an example method 1100 for automatically (or substantially automatically) performing a first real-time assay on a sample using system 1000. In this example, the assay is a production titer, and method 1100 generally includes: (1) moving (e.g., automatically) a needle to obtain a sample from one of the sample vials and place the sample in a sample loop, (2) forcing the sample from the sample loop into a needle seat port, (3) moving the sample from the needle seat port through multiport valve 104 and multiport valve 108 to an analytical device (in this example, taking the form of a liquid chromatography device), and (4) determining the titer / concentration of the sample using the analytical device.

[0066] 12 shows another example method 1200 for automatically (or substantially automatically) performing a second real-time assay on a sample using system 1000. In this example, the assay is a size exclusion chromatography (SEC) assay, and method 1200 generally includes: (1) moving (e.g., automatically) a needle to obtain a sample containing molecules from a first vial (one of the sample vials) and place the sample into a sample loop; (2) forcing the sample from the sample loop into a needle seat port; (3) moving the sample from the needle seat port through multiport valve 104 to multiport valve 108; (4) moving the sample from multiport valve 108 to a first column through multiport valve 112 (in a second position); and (5) capturing molecules in the sample in the first column, thereby separating the molecules in the sample from the matrix of the sample, which is separated from the matrix of the sample. (6) transferring an elution buffer solution from the buffer source to the first column via the needle, needle seat port, and multiport valves 104, 108, and 112, thereby eluting the molecules captured by the first column; (7) transferring an elution / molecule mixture comprising the elution buffer solution and the eluted molecules to a second vial (one of the receiving vials), which separates the molecules in the mixture from the remainder of the mixture; (8) transferring the molecules from the second vial to an analytical device via the needle, needle seat port, and multiport valves 104, 108; and (9) quantifying the level of aggregation in the molecules using the analytical device.

[0067] FIG. 13 illustrates another example method 1300 for automatically (or substantially automatically) performing a third real-time assay on a sample using system 1000.In this example, the assay is an ion exchange chromatography (IEX) assay, and method 1300 generally includes: (1) moving (e.g., automatically moving) a needle to obtain a sample (where the sample contains molecules) from a first vial (one of the sample vials) and place the sample into a sample loop; (2) forcing the sample from the sample loop into a needle seat port; (3) moving the sample from the needle seat port through multiport valve 104 to multiport valve 108; and (4) discharging the sample from multiport valve 108 to a first column through multiport valve 112 (a second column). (5) capturing molecules in the sample in the first column, thereby separating the molecules in the sample from the sample matrix, which flows out of the first column and is fluidly connected to the first column to waste; (6) transferring an elution buffer solution from the buffer source to the first column through the needle, needle seat port, and multiport valves 104, 108, and 112, thereby eluting the molecules captured by the first column; and (7) transferring an elution / molecule mixture comprising the elution buffer solution and the eluted molecules to a second column. (8) transferring the molecules from the second vial to a third vial (one of the intermediate vials) to dilute the affinity-purified sample; (9) transferring the affinity-purified sample from the third vial to multiport valve 108 via the needle, needle seat port, and multiport valve 104; and (10) transferring the affinity-purified sample from multiport valve 108 to a second column via the multiport valve. (11) applying the affinity purified sample to a second column, thereby further reducing the salt concentration of the sample; (12) transferring this further desalted affinity purified sample from the second column back to the second vial; (13) transferring the affinity purified sample from the second vial to an analytical device via the needle, needle seat port, and multiport valves 104, 108; and (14) performing charge variant assessment using the analytical device (which in this example takes the form of an ion exchange column).

[0068] 14 illustrates another example method 1400 for automatically (or substantially automatically) performing a fourth real-time assay on a sample using system 1000. In this example, the assay is a MAM assay, and method 1400 generally includes: (1) moving (e.g., automatically) a needle to obtain a sample from a first vial (one of the sample vials) and place the sample in a sample loop; (2) forcing the sample from the sample loop into a needle seat port; (3) moving the sample from the needle seat port through multiport valve 104 to multiport valve 108; and (4) discharging the sample from multiport valve 108 to a first column through multiport valve 112 (in a second position). (5) capturing molecules in the sample in the first column, thereby separating the molecules in the sample from the sample matrix, which flows out of the first column and is fluidly connected to the first column and disposed of; (6) transferring an elution buffer solution from the buffer source to the first column through the needle, needle seat port, and multiport valves 104, 108, and 112, thereby eluting the molecules captured by the first column; and (7) transferring the elution buffer solution and the eluted molecules from the buffer source to the first column through the needle, needle seat port, and multiport valves 104, 108, and 112, thereby eluting the molecules captured by the first column. (8) transferring the elution / molecule mixture containing the denaturing reagent and the reducing reagent to a second vial (one of the receiving vials), which separates the molecules in the mixture from the remainder of the mixture, thereby producing an affinity-purified sample in the second vial; (9) transferring the affinity-purified sample from the second vial to a third vial (one of the intermediate vials) via a needle to dilute the affinity-purified sample; (10) transferring the diluted affinity-purified sample from the third vial back to the second vial via a needle; and (11) transferring the denaturing reagent and the reducing reagent to the second vial. (11) transferring the affinity purified sample, denaturing reagent, and reducing reagent from the second vial to the reaction coil via the needle, multiport valves 104, 108, and multiport valve 112 (in the fourth position); (12) incubating the mixture in the reaction coil, thereby denaturing and reducing the molecules; and (13) transferring the denatured and reduced molecules from the reaction coil to a fourth vial (another one of the receiving vials) fluidically connected to the reaction coil.(14) transferring the alkylating reagent to the fourth vial via the needle; (15) transferring the modified and reduced molecule and the alkylating reagent from the fourth vial to the reaction coil via the needle, multiport valves 104, 108, and multiport valve 112 (which is in the fourth position); (16) incubating the modified and reduced molecule and the alkylating reagent in the reaction coil, thereby alkylating the modified and reduced molecule; (17) transferring the modified, reduced, and alkylated molecule from the reaction coil to the fourth vial; (18) transferring the modified, reduced, and alkylated molecule from the fourth vial to the second column via the needle, needle seat port, multiport valves 104, 108, and multiport valve 112 (which is moved to the third position); (19) transferring the modified, reduced, and alkylated molecule. to the second column, thereby reducing the salt concentration of the molecules; (20) returning the desalted molecules from the second column to the second vial; (21) (before step (20) or after step (20)) transferring the enzyme to the second vial via the needle; (22) transferring the desalted molecules and enzyme from the second vial to a reaction coil via the needle, multiport valves 104, 108, and multiport valve 112 (moved to the fourth position); (23) incubating the desalted molecules and enzyme in the reaction coil, thereby digesting the molecules; (24) returning the digested molecules from the reaction coil to the fourth vial; (25) transferring the digested molecules from the fourth vial to an analytical device via the needle, needle seat port, and multiport valves 104, 108; and (26) analyzing the digested molecules using the analytical device.

[0069] 15 partially illustrates another example method 1500 for automatically performing a fifth real-time assay on a sample using system 1000. In this example, the assay is a non-chromatographic assay, and method 1500 generally includes: (1) moving (e.g., automatically moving) a needle to obtain a sample from a first vial (one of the sample vials) and place the sample into a sample loop; (2) forcing the sample from the sample loop into a needle seat port; (3) moving the sample from the needle seat port through multiport valve 104 to multiport valve 108; (4) moving the sample from multiport valve 108 to a second vial (one of the receiving vials) through multiport valves 108, 112; (5) moving a quench reagent into the second vial via the needle; and (6) discharging the sample and quench reagent into the second vial. (7) transferring the quenched molecules from the vial to the reaction coil via the needle, multiport valves 104, 108, and multiport valve 112 (in the fourth position); (7) incubating the sample and quenching reagent in the reaction coil, thereby quenching the reaction of the molecules in the sample; (8) returning the quenched molecules from the reaction coil to a second vial or a third vial (another one of the receiving vials) fluidically connected to the reaction coil; (9) transferring the quenched molecules from the third vial to an analytical device via the needle, needle seat port, and multiport valves 104, 108; and (10) analyzing the quenched molecules using the analytical device.

[0070] 16 and 17 illustrate an example of a flow-through vial 1600 that can be used in system 100 or system 1000 to enable continuous flow of fluid through system 100 or system 1000. As shown, flow-through vial 1600 in this example generally includes a base 1604, a lip 1606 connected to base 1604, and a gutter 1608 connected to both base 1604 and lip 1606 and extending outwardly therefrom. In this example, base 1604 is defined by a first substantially cylindrical portion 1612 and a second substantially rectangular portion 1616 connected to first substantially cylindrical portion 1612. First substantially cylindrical portion 1612 generally extends along a first axis 1628 from a first end 1620 to a second end 1624 opposite first end 1620. First substantially cylindrical portion 1612 has a substantially cylindrical bore 1632 formed between first end 1620 and second end 1624 thereof. Second substantially rectangular portion 1616 extends outward from first substantially cylindrical portion 1612 along a second axis 1636 perpendicular to first axis 1628. However, in other examples, the components of the flow-through vial may have different shapes. For example, gutter 1608 may have a cylindrical or other non-rectangular shape. As another example, base 1604 may have a cylindrical or substantially cylindrical shape defined only by substantially cylindrical portion 1612.

[0071] In this example, lip 1606 is a substantially cylindrical lip that is integrally formed with first substantially cylindrical portion 1612 (particularly, first end 1620 thereof) and extends outward from first substantially cylindrical portion 1612 along first axis 1628. So positioned, lip 1606 fluidly connects substantially cylindrical bore 1632 to gutter 1608. However, in other examples, lip 1606 can have a different shape, can be fixedly or removably coupled to first substantially cylindrical portion 1612 (or another portion), and / or can extend outward from first substantially cylindrical portion 1612 along an axis that is angled relative to first axis 1628. In still other examples, flow-through vial 1600 may not include lip 1606 at all, in which case gutter 1608 is directly fluidly connected to substantially cylindrical bore 1632.

[0072] In this example, the gutter 1608 is defined by a substantially rectangular base 1640 and a downspout 1644 coupled to the rectangular base 1640. The substantially rectangular base 1640 of the gutter 1608 extends from the second substantially rectangular portion 1616 of the base 1604 and outward from the lip 1606 along a third axis 1648 that is perpendicular to both the first axis 1628 and the second axis 1636 and is inclined relative to the horizontal (see FIG. 17 ). The substantially rectangular base 1640 defines a substantially rectangular channel 1652 that fluidly connects the lip 1606 (and the subsequent substantially cylindrical bore 1632) to the downspout 1644. The downspout 1644 in this example is cylindrical and extends outward (downward in FIG. 16 ) from the rectangular base 1640 along a fourth axis 1652 that is parallel to the first axis 1628 and perpendicular to the second axis 1636 and the third axis 1648. In other examples, the gutter 1608 can have a base 1640 with a different shape (e.g., the base 1640 can have a cylindrical shape) and / or the downspout 1644 can have a different shape. In other examples, the gutter 1608 may not include a downspout 1644.

[0073] Flow-through vial 1600 also has an inlet port 1660 and an outlet port 1664. Inlet port 1660 is generally located in base 1604 of the flow-through vial to receive input fluids as needed from an automated sampling system, multiport valve 112, multiport valve 116, or other components of system 100 or system 1000. As shown in FIGS. 16 and 17 , inlet port 1660 in this example is formed in second substantially rectangular portion 1616 and extends along a fifth axis 1668 that is inclined relative to the orientation of first axis 1628, second axis 1636, third axis 1648, and fourth axis 1652. Fifth axis 1668 may be oriented at an angle of approximately 45 degrees relative to first axis 1628, for example. Meanwhile, outlet port 1664 is generally positioned in the gutter 1608 of the flow-through vial to direct fluid that enters base 1604 via inlet port 1660 and exits flow-through vial 1600 through substantially cylindrical bore 1632 and channel 1652 of gutter 1608. In this example, outlet port 1664 is formed in downspout 1644, although in other examples (e.g., when flow-through vial 1600 does not include downspout 1644), outlet port 1664 can be formed in a different portion of gutter 1608.

[0074] Although not shown here, it will be understood that a conduit (not shown) can be used to fluidly connect a desired component of system 100 (e.g., an automated sampling system) or system 1000 to inlet port 1660. Fluid then flows from the desired component of the system through inlet port 1660 and into flow-through vial 1600. Lip 1606 helps prevent surface tension as the fluid flows through flow-through vial 1600. In some examples, a conduit (not shown) can also be used to fluidly connect outlet port 1664 to a desired destination for the fluid within system 100 or system 1000. As an example, a conduit can be used to fluidly connect outlet port 1664 to an analytical device of system 100 or system 1000. In other examples, a conduit need not be used to fluidly connect outlet port 1664 to a desired destination. For example, a needle of system 100 or system 1000 can draw fluid directly from flow-through vial 1600 and place the fluid into a desired component of system 100 (e.g., a sample loop) or system 1000. As another example (e.g., if flow-through vial 1600 needs to be washed), flow-through vial 1600 can be positioned such that downspout 1644 sits directly over waste, such that fluid flowing through gutter 1608 is directed to waste. In either case, flow-through vial 1600 is configured to facilitate continuous flow therethrough, thereby eliminating the need for, for example, periodic replacement of sample vials (especially when different samples are being tested).

[0075] 18 and 19 show a vial holder 1800 that can be used in system 100 or system 1000 to hold a plurality of flow-through vials 1600. In this example, vial holder 1800 is configured to be disposed within or otherwise coupled to an automated sampling system. However, in other examples, vial holder 1800 can be coupled to another component of system 100 or system 1000. In either case, vial holder 1800 shown in FIGS. 18 and 19 includes a mounting flange 1804 and a plurality of vial receptacles 1808 coupled to mounting flange 1804. As shown in FIG. 19, mounting flange 1804 can be coupled to a portion of an automated sampling system to couple vial holder 1800 (and flow-through vials 1600 carried by vial holder 1800) to the automated sampling system. 19, each of the plurality of vial receptacles 1808 is sized to receive one of the flow-through vials 1600. More specifically, as shown in FIG. 19, each of the plurality of vial receptacles 1808 is sized to receive a respective base 1604 of one of the flow-through vials 1600. The gutter 1608 of each of the flow-through vials 1600 then extends outward, away from the vial holder 1800.

[0076] Therapeutic Polypeptides Proteins may be useful in the disclosed systems and methods, including proteins that bind to one or more of the following: CD proteins, including CD3, CD4, CD8, CD19, CD20, CD22, CD30, and CD34, including those that disrupt receptor binding; HER receptor family proteins, including HER2, HER3, HER4, and EGF receptor; cell adhesion molecules, such as LFA-I, MoI, p150, 95, VLA-4, ICAM-I, VCAM, and alpha v / beta 3 integrin. Growth factors, such as vascular endothelial growth factor ("VEGF"), growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, Müllerian inhibitory substance, human macrophage inflammatory protein (MIP-I-alpha), erythropoietin (EPO), nerve growth factors such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors including, for example, aFGF and bFGF, epidermal growth factor (EGF), transforming growth factors (TGFs), particularly TGF-α and TGF-β, for example, TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5, insulin-like growth factor I and insulin-like growth factor II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), and bone morphogenetic factors. Insulin and insulin-related proteins, e.g., insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor binding proteins; coagulation and coagulation-related proteins, such as factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators, e.g., urokinase and tissue plasminogen activator ("t-PA"), bombazine, thrombin, and thrombopoietin; (vii) other blood and serum proteins, including, but not limited to, albumin, IgE, and blood group antigens; colony-stimulating factors and their receptors, including, in particular, M-CSF, GM-CSF, and G-CSF, and their receptors, such as the CSF-1 receptor (c-fms).Receptors and receptor-associated proteins, including, for example, flk2 / flt3 receptor, obesity (OB) receptor, LDL receptor, growth hormone receptor, thrombopoietin receptor ("TPO-R", "c-mpl"), glucagon receptor, interleukin receptor, interferon receptor, T cell receptor, stem cell factor receptor such as c-Kit, and other receptors. Receptor ligands, including, for example, OX40L, which is a ligand for the OX40 receptor. Neurotrophic factors, including bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6). Relaxin A chain, relaxin B chain, and prorelaxin; interferons and interferon receptors, including, for example, interferon-α, -β, and -γ, and their receptors. Interleukins and interleukin receptors, including IL-1 through IL-33 and IL-1 through IL-33 receptors, such as, in particular, IL-8 receptor. Viral antigens, including AIDS envelope viral antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor alpha and beta, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-related peptide, DNAse, inhibin, and activin, integrins, protein A or D, rheumatoid factor, immunotoxins, bone morphogenetic proteins (BMPs), superoxide dismutase, surface membrane proteins, decay-accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, addressins, regulatory proteins, immunoadhesins, and antibodies. Myostatin, TALL proteins including TALL-I, amyloid proteins including but not limited to amyloid beta protein, thymic stromal lymphopoietin ("TSLP"), RANK ligand ("OPGL"), c-kit, TNF receptors including TNF receptor type 1, TRAIL-R2, angiopoietin, and biologically active fragments or analogs or variants of any of the foregoing.

[0077] Exemplary polypeptides and antibodies include Activase® (alteplase); alirocumab, Aranesp® (darbepoetin alfa), Epogen® (epoetin alfa, or erythropoietin); Avonex® (interferon beta-Ia); Bexxar® (tositumomab); Betaseron® (interferon beta); bococizumab (anti-PCSK9 monoclonal antibody designated as L1L3, see U.S. Pat. No. 8,080,243); Campath® (alemtuzumab); Dynepo® (epoetin delta); Velcade® (bortezomib); MLN0002 (anti-α4β7 mAb); MLN1202 (anti-CCR2 chemokine receptor mAb); Enbrel® (etanercept); Eprex® (epoetin alfa); Erbitux® (cetuximab); evolocumab; Genotropin® (somatropin); Herceptin® (trastuzumab); Humatrope® (somatropin [rDNA-derived] injection); Humira® (adalimumab); Infergen® (interferon Alfacon-1); Natrecor® (nesiritide); Kineret® (anakinra), Leukine® (sargamostim); Lympho Cide® (epratuzumab); Benlysta™ (belimumab); Metalyse® (tenecteplase); Mircera® (methoxypolyethylene glycol epoetin beta); MyIotarg® (gemtuzumab ozogamicin); Raptiva® (efalizumab); Cimzia® (certolizumab pegol); Soliris™ (eculizumab); pexelizumab (anti-complement C5); MEDI-524 (Numax®); Lucentis® (ranibizumab); edrecolomab (Panorex®); Trabio® (lerdelimumab); TheraCim hR3 (nimotuzumab); Omnitarg (pertuzumab, 2C4);Osidem® (IDM-I); OvaRex® (B43.13); Nuvion® (vigilizumab); cantuzumab mertansine (huC242-DMl); NeoRecormon® (epoetin beta); Neumega® (oprelvekin); Neulasta® (PEGylated filgastrim, PEGylated G-CSF, PEGylated hu-Met-G-CSF); Neupogen® (filgrastim); Orthoclone OKT3® (muromonab-CD3), Procrit® (epoetin alfa); Remicade® (infliximab), Reopro® (abciximab), Actemra® (anti-IL6 receptor mAb), Avastin® (bevacizumab), HuMax-CD4 (zanolimumab), Rituxan® (rituximab); Tarceva® (erlotinib); Roferon-A® (interferon alfa-2a); Simulect® ®) (basiliximab); Stelara™ (ustekinumab); Prexige® (lumiracoxib); Synagis® (palivizumab); 146B7-CHO (anti-IL15 antibody, see U.S. Pat. No. 7,153,507), Tysabri® (natalizumab); Valortim® (MDX-1303, anti-anthrax protective antigen mAb); ABthrax™; Vectibix® (panitumumab); Xolair® (omalizumab), ETI211 (anti-MRSA) mAb), IL-1 Trap (the Fc portion of human IgG1 and the extracellular domains of both IL-1 receptor components (type I receptor and receptor accessory protein)), VEGF Trap (the Ig domain of VEGFR1 fused to IgG1 Fc), Zenapax® (daclizumab); Zenapax® (daclizumab), Zevalin® (ibritumomab tiuxetan), Zetia (ezetimibe), atacicept (TACI-Ig), anti-α4β7 mAb (vedolizumab); galiximab (anti-CD80 monoclonal antibody), anti-CD23 mAb (lumiliximab);BR2-Fc (huBR3 / huFc fusion protein, soluble BAFF antagonist); Simponi™ (golimumab); mapatuzumab (human anti-TRAIL receptor-1 mAb); ocrelizumab (anti-CD20 human mAb); HuMax-EGFR (zalutumumab); M200 (volociximab, anti-α5β1 integrin mAb); MDX-010 (ipilimumab, anti-CTLA-4 mAb and VEGFR-I (IMC-18F1); anti-BR3 mAb; anti-C. difficile toxin A and toxin BC mAbs MDX-066 (CDA-1) and MDX-1388); anti-CD22 dsFv-PE38 conjugate (CAT-3888 and CAT-8015); anti-CD25 mAb (HuMax-TAC); anti-TSLP antibody; anti-TSLP receptor antibody (U.S. Pat. No. 8,101,182); anti-TSLP antibody designated as A5 (U.S. Pat. No. 7,982,016); anti-CD3 mAb (NI-0401); adecatumumab (MT201, anti-EpCAM-CD326 mAb); MDX-060, SGN-30, SGN-35 (anti-CD30 mAb); MDX-1333 (anti-IFNAR); HuMax CD38 (anti-CD38 mAb); anti-CD40L mAb; anti-Cripto mAb; anti-CTGF idiopathic pulmonary fibrosis stage 1 fibrogen (FG-3019); anti-CTLA4 mAb; anti-eotaxin 1 mAb (CAT-213); anti-FGF8 mAb; anti-ganglioside GD2 mAb; anti-sclerostin antibody (see U.S. Patent No. 8,715,663 or U.S. Patent No. 7,592,429), anti-sclerostin antibody designated as Ab-5 (see U.S. Patent No. 8,715,663 or U.S. Patent No. 7,592,429); anti-ganglioside GM2 mAb; anti-GDF-8 human mAb (MYO-029); anti-GM-CSF receptor mAb (CAM-3001); anti-HepC mAb (HuMax HepC); MEDI-545, MDX-1103 (anti-IFNα mAb); anti-IGF1R mAb; anti-IGF-1R mAb (HuMax-Inflam); anti-IL12 / IL23p40 mAb (briakinumab); anti-IL-23p19 mAb (LY2525623); anti-IL13 mAb (CAT-354); anti-IL-17 monoclonal antibody (AIN457); anti-IL2Ra mAb (HuMax-TAC);Examples of such antibodies include anti-IL5 receptor mAb; anti-integrin receptor mAb (MDX-O18, CNTO95); anti-IPIO ulcerative colitis mAb (MDX-1100); anti-LLY antibody; BMS-66513; anti-mannose receptor / hCGβ mAb (MDX-1307); anti-mesothelin dsFv-PE38 conjugate (CAT-5001); anti-PDlmAb (MDX-1 106 (ONO-4538)); anti-PDGFRα antibody (IMC-3G3); anti-TGFβ mAb (GC-1008); anti-TRAIL receptor-2 human mAb (HGS-ETR2); anti-TWEAK mAb; anti-VEGFR / Flt-1 mAb; anti-ZP3 mAb (HuMax-ZP3); and amyloid beta monoclonal antibodies comprising the sequences of SEQ ID NO:8 and SEQ ID NO:6 (U.S. Patent No. 7,906,625).

[0078] Examples of antibodies suitable for the methods and pharmaceutical formulations include those shown in Table 1. Other examples of suitable antibodies include infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, acelizumab, altinumab, atlizumab, atolimu- umab, bapineuzumab, basilisk, basiliki ... Cimab, bavituximab, bectumomab, belimumab, benralizumab, bertilimumab, besilesomab, bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab, certolizumab pegol, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clinol Batuzumab tetraxetan, conatumumab, crenezumab, cr6261, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab alitox, drozitumab, durigotumab, dupilumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, elotuzumab, ersilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erli Ibuprofen, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exibirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, framvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, furanumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin,Golimumab, gomiliximab, gs6624, ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab, imgatuzumab, inlacumab, indatuximab ravtansine, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab, remaresomab, lerdelimumab, lexatumumab, ribivirumab, ligelizumab, lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab, lumirixi Mab, mapatuzumab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minletumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-cd3, nacolomab butafenatox, namilumab, naptumomab estafenatox, narunatumumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, or Latumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pasivaximab, panitumumab, panobacumab, palsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pintumomab, placumab, ponezumab, priliximab, pritumumab, PRO140, quilizumab, racotumomab, radletuzumab, rafivirumab, ramucil Mab, raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab, lobatumumab, loredumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, samarizumab, sarilumab, satumomab pendetide, secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, subizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab,Taplitumomab paptox, tefibazumab, terimomab alitox, tenatumomab, teneliximab, teplizumab, teprotumumab, tezepelumab, TGN1412, tremelimumab, ticilimumab, tildrakizumab, tigatuzumab, TNX-650, tocilizumab, toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tucotu These include cermoleukin, tubilumab, ublituximab, urelumab, urtoxazumab, ustekinumab, bapaliximab, batelizumab, vedolizumab, veltuzumab, bepalimomab, besencumab, visilizumab, volociximab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, diralimumab, and zolimomab alitoxin.

[0079] Antibodies also include adalimumab, bevacizumab, blinatumomab, cetuximab, conatumumab, denosumab, eculizumab, erenumab, evolocumab, infliximab, natalizumab, panitumumab, rilotumumab, rituximab, romosozumab, tezepelumab, and trastuzumab, and antibodies selected from Table 1.

[0080] [Table 1]

[0081] [Table 2]

[0082] Based on the foregoing, it will be appreciated that the devices, systems, and methods described herein facilitate the automatic (or substantially automatic) preparation of a molecule-containing product sample for analysis and the automatic (or substantially automatic) performance of an assay on that sample. As used herein, "automatic" has its ordinary and accustomed meaning as would be understood by one of ordinary skill in the art in light of this disclosure. It refers to performing a process without human intervention during the process. An automated process may be performed by one or more machines. As used herein, "substantially automatic" has its ordinary and accustomed meaning as would be understood by one of ordinary skill in the art in light of this disclosure. It refers to performing a process in which more active steps are performed without human intervention than with human intervention. If additional numerical information is important, a process can be considered "substantially automatic" if at least 51%, 60%, 70%, 80%, or 90% of the process steps are performed without human intervention. For example, a process that is performed during a single day and involves 0.5 to 1 hour of "staff hand time" during the day can be considered substantially automatic. As used herein, "continuous" has its ordinary and accustomed meaning as would be understood by one of ordinary skill in the art in light of the present disclosure. It refers to performing at least two cycles of a process without interruption. The at least two cycles may be performed sequentially (e.g., one after the other) or overlapping. Thus, preparation and analysis can be performed in substantially real time. In other words, the entire process can be performed much more rapidly than currently possible with conventional processes.

[0083] Furthermore, applicants have found that assays performed using the systems and methods described herein produce results comparable to those produced by assays performed using conventional techniques, thereby confirming the effectiveness of the systems and methods described herein. Specifically, applicants performed three different assays (size-exclusion chromatography (SEC) assay, cation-exchange chromatography (CEX) assay, and reduced capillary electrophoresis (rCE) assay) using nine different molecules (molecules mAb1, mAb2, mAb3, BiTE1, BiTE2, bispecific1, fusion, bispecific2, and xmAb) using both the systems and methods described herein and conventional techniques. While many conventional techniques exist for sample affinity purification (e.g., PhyNexus Phy Tips, GE PreDictor plates, Hamilton Leap autosamplers, and Tecan-Atoll), Tecan-Atoll purification was used in this example as the conventional technique, which is considered representative of the prior art. As shown in Figure 20, Applicant found that the results of 25 of the 27 different assays were comparable to the results of the same 27 assays performed using conventional techniques, as indicated by the 25 different check marks. The remaining two assays could not be compared because no results were available from the conventional techniques. As used herein, "comparable" has its ordinary and accustomed meaning as would be understood by one of ordinary skill in the art in light of the present disclosure. For example, it was observed herein that the major peaks obtained by the various assays performed with the disclosed systems and methods were within 5% of the major peaks obtained by the various assays performed using conventional techniques, which would be understood to result in "comparable" results to conventional techniques.

[0084] 21 and 22 are graphs showing some of the results supporting Applicant's findings. Specifically, FIGS. 21 and 22 are graphs showing the results of rCE assays performed with one bispecific molecule using the systems and methods described herein and conventional techniques, respectively. As shown, the rCE assay performed with one bispecific molecule using the systems and methods described herein produced a Peak A value of 15.450 and a Peak B value of 19.658, while the rCE assay performed with one bispecific molecule using conventional techniques produced a Peak A value of 15.183 and a Peak B value of 19.458.

[0085] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. While many examples have been shown and described herein, those skilled in the art will readily understand that the details of various embodiments are not necessarily mutually exclusive. Rather, given the teachings herein, those skilled in the art will be able to combine one or more features of one embodiment with one or more features of the remaining embodiments. It will further be understood that the illustrated embodiments are merely exemplary and should not be construed as limiting the scope of the present disclosure. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any example or exemplary language (e.g., "such as") herein is intended merely to further clarify aspects or embodiments of exemplary embodiments of the present disclosure, and does not limit the scope of the disclosure. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

Claims

1. (a) transferring a sample containing molecules from a first vial to a sample loop; (b) transferring a volume of the sample from the sample loop to a first multiport valve; (c) transferring the aliquot of sample from the first multiport valve to a second multiport valve fluidly connected to and positioned downstream of the first multiport valve; (d) transferring the aliquot of sample from a second multiport valve to a trapping column; (e) capturing the molecules in the aliquot of sample on the capture column, thereby separating the molecules in the sample from the sample matrix; (f) transferring a solution containing a glycosidase to the capture column through a second multiport valve to release glycans from molecules bound to the capture column; (g) transferring the released glycans from the capture column to a second vial disposed downstream of the capture column, the second vial comprising a flow-through vial; (h) mixing the released glycans with a glycan labeling reagent in the second vial; (i) transferring the mixture of the released glycans and the glycan labeling reagent to a reaction coil through the first multiport valve; (j) incubating a mixture of the released glycans and the glycan labeling reagent in a reaction coil, thereby labeling the glycans; (k) transferring the mixture from the reaction coil to a third vial, wherein the third vial comprises a flow-through vial; (l) transferring the labeled glycans in the third vial to an analytical device for analysis of the labeled glycans; A method comprising:

2. 2. The method of claim 1, wherein (h) comprises fluorescently labeling the glycans with the glycan labeling reagent.

3. 3. The method of claim 1 or 2, further comprising maintaining the reaction coil at a predetermined incubation temperature, wherein the predetermined temperature comprises 80 degrees Celsius.

4. 4. The method of claim 1, wherein (a) comprises automatically transferring the sample from the first vial to the sample loop with a needle.

5. The method of any one of claims 1 to 4, further comprising, before (a), automatically placing the sample into the first vial.

6. 6. The method of any one of claims 1 to 5, wherein one or more of (a) through (h) are performed automatically using a controller, and wherein (a) through (h) are performed in a closed system.

7. a first vial adapted to contain a sample containing the molecule; a sample loop adapted to receive the sample from the first vial; a first multiport valve fluidly connected to the sample loop and configured to obtain a volume of the sample through a first port of the first multiport valve; a second multiport valve fluidly connected to the first multiport valve and disposed downstream of the first multiport valve; a capture column disposed in fluid communication with the second multiport valve when the second multiport valve is in a first position, the capture column configured to capture the molecules from the sample; a second vial located downstream of the capture column, the second vial being a flow-through vial and containing a glycan labeling reagent; a glycosidase source configured to supply the glycosidase to the capture column through the second multiport valve so as to contact the glycosidase with the molecule bound to the capture column; a carrier solution source positioned to supply a carrier solution to the capture column through the second multiport valve, the carrier solution releasing glycans from the capture column and transporting them to the second vial; a reaction coil configured to receive a mixture of the released glycans and the glycan labeling reagent from the second vial through the first multiport valve and to incubate the mixture of the released glycans and the glycan labeling reagent to label the glycans; a third vial positioned downstream of the reaction coil for receiving the labeled glycan and the glycan labeling reagent; A closure system including:

8. 8. The closed system of claim 7, further comprising a needle configured to automatically withdraw the sample from the first vial and place the sample in the sample loop.

9. 9. The closed system of claim 7 or 8, further comprising a heating element positioned immediately adjacent to the reaction coil, the heating element configured to maintain the reaction coil at a temperature of 80 degrees Celsius.

10. The method of any one of claims 1 to 6, wherein the glycosidase is selected from the group consisting of endoglycosidases, glycosamidases, and O-glycanases, and combinations thereof.

11. The method of any one of claims 1 to 6, wherein the glycan labeling reagent is a fluorophore or a chromophore.

12. The method of any one of claims 1 to 6, wherein the molecule comprises a polypeptide and the capture column is a polypeptide-binding column.

13. 13. The method of claim 12, wherein the polypeptide binding column is selected from the group consisting of a Protein A column, a Protein G column, a Protein A / G column, a Protein L column, an amino acid column, an avidin column, a streptavidin column, a carbohydrate binding column, a carbohydrate column, a glutathione column, a heparin column, a hydrophobic interaction column, an immunoaffinity column, a nucleotide / coenzyme column, a specialty column, and an immobilized metal affinity chromatography (IMAC) column.

14. 7. The method of any one of claims 1 to 6, wherein the flow-through vial comprises a base, a gutter coupled to and extending outwardly from the base, an inlet port formed in the base, and an outlet port formed in the gutter.

15. The method of any one of claims 1 to 6, wherein the third vial is also a flow-through vial configured to substantially filter the glycan labeling reagent from the third vial, thereby leaving substantially only labeled glycans in the third vial.

16. A closed system described in any one of claims 7 to 9, wherein the glycosidase is selected from the group consisting of endoglycosidases, glycosamidases, and O-glycanases, and combinations thereof.

17. A closed system described in any one of claims 7 to 9 and 16, wherein the glycan labeling reagent is a fluorophore or chromophore.

18. A closed system described in any one of claims 7 to 9, 16, and 17, wherein the molecule comprises a polypeptide and the capture column is a polypeptide binding column.

19. The closed system of claim 18, wherein the polypeptide binding column is selected from the group consisting of a protein A column, a protein G column, a protein A / G column, a protein L column, an amino acid column, an avidin column, a streptavidin column, a carbohydrate binding column, a carbohydrate column, a glutathione column, a heparin column, a hydrophobic interaction column, an immunoaffinity column, a nucleotide / coenzyme column, a specialty column, and an immobilized metal affinity chromatography (IMAC) column.

20. A closed system as described in any one of claims 7 to 9 and 16 to 19, wherein the flow-through vial includes a base, a gutter connected to the base and extending outward from the base, an inlet port formed in the base, and an outlet port formed in the gutter.

21. A closed system described in any one of claims 7 to 9 and 16 to 20, wherein the third vial is also a flow-through vial configured to substantially filter the glycan labeling reagent from the third vial, thereby leaving substantially only the labeled glycan in the third vial.

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