Liquid chromatography-mass spectroscopy (LC-ms) method for analyzing fluctuation of ampholyte lot

Abstract

To provide a method for characterizing a protein composition using charges, a method for characterizing an ampholyte composition using liquid chromatography-mass spectroscopy (LS-MS), and an improved method for directly analyzing an ampholyte composition using CIEF and iCIEF.SOLUTION: A method comprises: (a) using LC-MS to identify markers in a test ampholyte composition and in a reference ampholyte composition; and (b) determining the degree of similarity or difference between the markers in the test ampholyte composition and the reference ampholyte composition.SELECTED DRAWING: Figure 4A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 913,450, filed October 10, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Technical Field The present disclosure relates to methods for characterizing protein compositions by charge and for characterizing ampholyte compositions using liquid chromatography-mass spectrometry (LC-MS). [Background technology]

[0003] background Many proteins undergo secondary modifications that result in protein charge variations, such as deamidation, formation of N-terminal pyroglutamic acid, aggregation, isomerization, sialylated glycans, fragmentation, and glycosylation at lysine residues. In some cases, these secondary modifications and resulting charge variants can affect binding, biological activity, patient safety, and protein shelf life. Protein charge can be analyzed using tools such as isoelectric focusing gel electrophoresis (IEF) and its capillary equivalents, such as capillary isoelectric focusing (CIEF) and imaged CIEF (iCIEF). These techniques are important for characterizing and monitoring the quality, purity, stability, and variability of therapeutic proteins, including protein-based drug products and active pharmaceutical ingredients. One such technique, iCIEF, has significantly contributed to biopharmaceutical development due to its high resolution, minimal development time, reduced sample volume, and fast run times. These advantages enable its application throughout the pharmaceutical process, from cell culture development and optimization to commercial quality control (QC) release and stability activities.

[0004] In CIEF and iCIEF, separation of proteins with different net charges is achieved by focusing proteins in an ampholyte pH gradient via an applied electric field and separating isoforms based on their inherent isoelectric points. This separation technique relies on ampholytes to create a pH gradient when placed under an electric potential. Protein species then migrate to different regions of the capillary based on their isoelectric points or pI values. However, lot-to-lot variation in the ampholyte composition used to generate the pH gradient in these methods can affect assay results. Previous methods for characterizing ampholyte compositions for compatibility with iCIEF and similar techniques have relied on indirect methods, such as using an ampholyte composition to generate an iCIEF electropherogram or similar readout of a reference protein and comparing the resulting electropherograms generated with different ampholyte compositions. However, these methods are expensive, time-consuming, and require large amounts of protein. Therefore, there is a need in the art for improved methods for characterizing ampholyte compositions for compatibility in methods such as iCIEF. The present invention fulfills this need by providing an improved method for directly analyzing ampholyte compositions. Summary of the Invention

[0005] overview The present disclosure provides a method for identifying a test ampholyte composition having an appropriate activity, the method comprising: (a) identifying at least one marker in at least one test ampholyte composition and one reference ampholyte composition using liquid chromatography-mass spectrometry (LC-MS); and (b) determining the degree of similarity or difference of the at least one marker between the at least one test ampholyte composition and the reference ampholyte composition, wherein the at least one test ampholyte composition has an appropriate activity if the at least one marker has a low covariance and the level of the at least one marker is different between the at least one test ampholyte composition and the reference ampholyte composition, or the at least one test ampholyte composition has an appropriate activity if the at least one marker has a high covariance and the level of the at least one marker is similar between the at least one test ampholyte composition and the reference ampholyte composition, thereby identifying a test ampholyte composition having an appropriate activity.

[0006] In some embodiments of the disclosed method, step (a) comprises: (i) determining accurate mass / retention time (AMRT) or collision cross section (CCS) measurements of a plurality of components of at least one test ampholyte composition and a reference ampholyte composition; (ii) using an S-plot to plot the covariance of the AMRT or collision cross section measurements of the plurality of components from the at least one test ampholyte composition and the reference ampholyte composition; and (iii) selecting at least one component that differs between the at least one test ampholyte composition and the reference ampholyte composition, wherein the difference comprises a covariance that is non-zero in the S-plot, thereby identifying at least one marker for characterizing the suitability of the at least one test ampholyte composition. In some embodiments, the difference comprises a covariance that is less than zero in the S-plot. In some embodiments, at least one component of the plurality of components in the S-plot comprises a covariance in the S-plot that is less than at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the covariance. In some embodiments, the at least one test ampholyte composition has suitable activity if the levels of the at least one marker in the at least one test ampholyte composition and the reference ampholyte composition differ.

[0007] In some embodiments of the disclosed methods, the difference in the level of at least one marker comprises a difference in level of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% relative to the normalized level of the at least one marker.

[0008] In some embodiments of the disclosed method, step (a) comprises: (i) determining accurate mass / retention time (AMRT) or collision cross section measurements of a plurality of components of at least one test ampholyte composition and a reference ampholyte composition; (ii) using an S-plot to plot the covariance of the AMRT or collision cross section measurements of the plurality of components from the at least one test ampholyte composition and the reference ampholyte composition; and (iii) selecting at least one component that is similar between the at least one test ampholyte composition and the reference ampholyte composition, where the similarity comprises a covariance that is non-zero in the S-plot, thereby identifying at least one marker for characterizing the suitability of the at least one test ampholyte composition. In some embodiments, the difference comprises a covariance that is greater than zero in the S-plot. In some embodiments, at least one component has a covariance in the S-plot that is greater than the covariance of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the components in the S-plot. In some embodiments, the at least one test ampholyte composition has suitable activity if the levels of at least one marker in the at least one test ampholyte composition and the reference ampholyte composition are similar.

[0009] In some embodiments of the disclosed methods, similarity in the level of at least one marker includes levels that are at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% similar compared to the normalized level of the at least one marker.

[0010] In some embodiments of the disclosed methods, at least one marker is identified by its mass-to-charge ratio (m / z). In some embodiments, the level of the at least one marker is characterized by the relative intensity of the m / z in the mass spectrum.

[0011] In some embodiments of the disclosed method, step (b) comprises: (i) determining an LC-MS mass spectrum of the at least one marker in the at least one test ampholyte composition and in the reference ampholyte composition; (ii) determining the relative intensity of a reference peak of the at least one marker in the mass spectra of the at least one test ampholyte composition and the reference ampholyte composition; (iii) normalizing the relative intensity of the reference peak of the at least one marker to the maximum relative intensity of the reference peak measured from the at least one test ampholyte composition or the reference ampholyte composition; and (iv) comparing the normalized relative intensity of the reference peak of the at least one marker in the at least one test ampholyte composition and the reference ampholyte composition.

[0012] In some embodiments of the disclosed methods, the at least one marker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 markers with different m / z. In some embodiments, the at least one marker comprises 16 markers with different m / z. In some embodiments, the 16 markers comprise markers at m / z 280, m / z 319, m / z 329, m / z 347, m / z 373, m / z 375, m / z 376, m / z 431, m / z 504, m / z 506, m / z 508, m / z 520, m / z 534, m / z 562, m / z 906, and m / z 980.

[0013] In some embodiments of the disclosed methods, the mass spectrometry comprises ion mobility quadrupole time-of-flight mass spectrometry (IMS-Q-ToF-MS).

[0014] In some embodiments of the disclosed methods, the liquid chromatography comprises high performance liquid chromatography (HPLC). In some embodiments, the HPLC comprises a C4 silica column.

[0015] In some embodiments of the disclosed methods, the method further includes validating the at least one test ampholyte composition by generating imaged capillary isoelectric focusing (iCIEF) electropherograms of the reference protein using the reference ampholyte composition and the at least one test ampholyte composition, thereby generating at least one test electropherogram and one reference electropherogram.

[0016] In some embodiments of the disclosed methods, the test ampholyte composition is verified for similarity of at least one test electropherogram and a reference electropherogram. In some embodiments, the similarity of the at least one test electropherogram and the reference electropherogram is determined by the number, size, or isoelectric point (pI) of peaks, or a combination thereof.

[0017] In some embodiments of the disclosed methods, the suitable activity comprises capillary isoelectric focusing (CIEF) or imaged capillary isoelectric focusing (iCIEF). In some embodiments, iCIEF is used to characterize a protein drug product or drug substance. [Brief explanation of the drawings]

[0018] [Figure 1]Figures 1A-1B are a pair of electropherograms analyzing a reference protein product using an imaged capillary isoelectric focusing (iCIEF) assay. The x-axis indicates time (in minutes) and the y-axis indicates absorbance. Figure 1A uses ampholyte lot 1. Figure 1B shows an exemplary candidate ampholyte lot, which produces a different electropherogram profile than the ampholyte lot shown in Figure 1A for the same reference protein. The differences in the electropherograms are circled. [Figure 2] Figure 1 shows a plot showing baseline interference due to the ampholyte gradient used in iCIEF. ICIEF was run without reference protein. Increasing ampholyte concentration led to a decrease in the baseline. This change in peak profile was due to ampholytes, not the protein or the instrument. [Figure 3] Electropherograms showing that pharmalyte (ampholyte) concentration results in a change in region area: a 2% change in pharmalyte concentration resulted in a 1% decrease in the area of Region 1. [Figure 4A] A series of electropherograms. Lots 7 and 2 are most similar to lot 1 and are the best candidates for lot statistical analysis. [Figure 4B] 1 is a table showing resolution differences within ampholyte lots. Lots 7 and 2 are most similar to lot 1 and are the best candidates for lot statistical analysis. [Figure 5A] Figure 5A is a series of plots showing isoelectric point (PI, or pI) marker testing of ampholyte lots. The peak resolution for each ampholyte lot was calculated to be 7.0-7.05. Figure 5A shows an example electropherogram with two peaks. [Figure 5B] Figure 5B is a series of plots showing isoelectric point (PI, or pI) marker testing of ampholyte lots. The peak resolution for each ampholyte lot was calculated to be between 7.0 and 7.05. Figure 5B shows how resolution is calculated from the electropherogram. Resolution is calculated based on migration (run) time (Rt) and peak width (W). [Figure 5C] Figure 5C shows a series of plots showing isoelectric point (PI, or pI) marker testing of ampholyte lots. The peak resolution for each ampholyte lot was calculated to be 7.0-7.05. Figure 5C shows an example electropherogram with 0.15% mutual overlap between peaks, or a resolution of 1.5, the minimum standard for chromatography. [Figure 6] FIG. 1 outlines the problem solved by the method of the present disclosure. [Figure 7] FIG. 1 shows the ampholyte LC-MS analytical workflow. [Figure 8] Figure 8A is a table showing GE Healthcare IEF carrier ampholyte lots tested for molecular composition. Figure 8B is a Waters S-plot showing testing of ampholyte lots for variation in molecular composition, rather than performance. Liquid chromatography-mass spectrometry (LC-MS) was used to identify components of interest in ampholyte lots with high lot-to-lot variation. Analysis was performed using UNIFI software with integrated data analysis and workflow. The S-plot shows the dissimilarity of accurate mass / retention time (AMRT) between ampholyte lots. AMRT pairs are plotted by covariance, with the magnitude of variation shown on the x-axis and the correlation, or consistency of variation, shown on the y-axis. [Figure 9] Figures 9A-9C are Waters S-plots comparing ampholyte lots characterized using Waters VION IMS QTof ion mobility quadrupole time-of-flight mass spectrometry (LC-IMS-Q-TOF-MS). Figure 9A shows a comparison of Lot 1 and Lot 2. Figure 9B shows a comparison of Lot 1 and Lot 3. Figure 9C shows a comparison of Lot 1 and Lot 5. Waters S-plots identify similarities and differences between samples and provide a powerful visual filtering tool. Unique features were selected for the library of ampholyte components, e.g., the highlighted points in the S-plot of Lot 1 vs. Lot 5 (Figure 9C). [Figure 10A]This table shows a library of ampholyte components characterized by m / z values (mass-to-charge ratios) and normalized relative intensities of ampholyte components in ampholyte lot numbers 1 (the current lot used in standard operating procedure, or SOP, lot), 2, 3, 4, and 5. Relative intensities were normalized to the highest relative intensity across all ampholyte lots. Marker response values were evaluated and compared in Excel. [Figure 10B] iCIEF electropherograms are shown for analyzing a Reference Protein 1 product using five different ampholyte lots. From top to bottom: Lot 1 (SOP), Lot 3, Lot 4, and Lot 5. Differences in the iCIEF profiles are circled. [Figure 10C] Shown are iCIEF electropherograms analyzing reference protein 1 product using ampholyte lots 1 (top, SOP) and 2 (bottom). Lot 2 was run after analyzing the LC-MS results and was predicted to be the lot most similar to lot 1 (see Figure 10A). [Figure 11] Figure 11A is a plot showing the ion mobility filtered extracted ion chromatogram (XIC) of marker m / z 906.261 from each lot of the indicated ampholyte. The XIC response is used for lot-to-lot comparisons. Figure 11B is a trend plot showing the marker in three replicates (x-axis indicates lot and replicate) of the indicated ampholyte lot versus marker intensity (y-axis, counts). Markers were considered relevant if they were identified in all three replicates of the current lot and were not detected in the blank. [Figure 12] The structures of three markers from the library used to characterize the ampholyte lots are shown. The structures correspond to the markers with the lower m / z values. These are the commercially available solutions. [Figure 13]This figure compares the current ampholyte testing process with the new ampholyte testing method described herein. Liquid chromatography-mass spectrometry (LC-MS) analysis of a new ampholyte lot takes 4 hours, so the current method reduces analysis time. Furthermore, the new ampholyte testing method allows for characterization of ampholytes without consuming the protein product. Finally, the new ampholyte testing method can be adapted to new ampholyte vendors. For each vendor, a new library of markers can be created using the method described here to compare ampholyte lots. DETAILED DESCRIPTION OF THE INVENTION

[0019] Detailed Description The present disclosure provides a method for identifying an ampholyte composition having an appropriate activity for a desired downstream application, the method comprising: (a) identifying at least one marker in at least one test ampholyte composition and one reference ampholyte composition using liquid chromatography-mass spectrometry (LC-MS); and (b) determining the degree of similarity or difference of the at least one marker between the at least one test ampholyte composition and the reference ampholyte composition. In some embodiments, the marker is selected based on the difference between the reference ampholyte composition and the test ampholyte composition, and if at least one marker is different between the at least one test ampholyte composition and the reference ampholyte composition, the at least one test ampholyte composition has the appropriate activity. In some embodiments, the marker is selected based on the similarity between the reference ampholyte composition and the test ampholyte composition, and if at least one marker is similar between the at least one test ampholyte composition and the reference ampholyte composition, the at least one test ampholyte composition has the appropriate activity. Markers can be identified by a particular mass-to-charge ratio (m / z) measured by mass spectrometry, and the level or marker can be determined by the relative intensity of the indicated m / z peak, which can optionally be normalized. This normalization can be to the maximum relative intensity measured for the indicated m / z peak across the ampholyte compositions in the analysis. Markers can be further characterized by their retention time in a liquid chromatography separation step.

[0020] Activities suitable for downstream applications include, but are not limited to, isoelectric focusing gel electrophoresis (IEF) and capillary equivalents such as capillary isoelectric focusing (CIEF) and imaged CIEF (iCIEF).

[0021] In some embodiments, the method further includes validating the at least one test ampholyte composition by generating imaging capillary isoelectric focusing (iCIEF) electropherograms of the reference protein using the reference ampholyte composition and the at least one test ampholyte composition, thereby generating at least one test electropherogram and one reference electropherogram. The similarity between the test and reference electropherograms can be determined by comparing the number, size, area, resolution, isoelectric point (pI), or a combination of all of these properties, of peaks in the reference and test electropherograms.

[0022] Use of ampholyte compositions The present disclosure provides methods for determining the suitability of an ampholyte composition for one or more downstream applications.

[0023] Ampholyte compositions, sometimes called pharmalytes (e.g., commercially manufactured ampholytes from GE Healthcare), are used in a variety of methods for separating proteins based on their isoelectric point. Exemplary methods for which the methods described herein can be used to optimize ampholyte compositions include, but are not limited to, isoelectric focusing gel electrophoresis (IEF) and capillary equivalents such as capillary isoelectric focusing (CIEF) and imaged CIEF (iCIEF).

[0024] As used herein, "ampholyte" refers to a compound containing both positive and negative charges that behaves as a zwitterion at or near its isoelectric point (pI). Ampholytes function as either an acid or a base depending on the pH of the solution into which they are introduced. Ampholytes aid in the formation of a pH gradient, for example, during focusing in iCIEF and CIEF.

[0025] The isoelectric point (pI) is the pH at which a molecule has no net charge. For proteins, the isoelectric point is the pH at which the overall charge of the protein is zero.

[0026] Isoelectric focusing gel electrophoresis (IEF) IEF is a technique for separating proteins based on charge, which involves adding an ampholyte solution to an immobilized pH gradient (IPG) gel. IPG consists of an acrylamide gel matrix copolymerized with a pH gradient, which can be generated by embedding carrier ampholytes in the acrylamide matrix. The resulting pH gradient is stable in an electric field except at highly alkaline (>12) pH values. The pH gradient for protein separation is fully established prior to the addition of protein analytes by first electrophoresing solutions of small molecules, such as polyampholytes, with varying pI values.

[0027] When an electric field is applied, proteins in the pH region below their isoelectric point (pI) are positively charged and therefore migrate toward the cathode (negatively charged electrode). As they move through the increasing pH gradient, the protein's overall charge decreases until the protein reaches the pH region corresponding to its pI. At this point, there is no net charge, so migration stops (because there is no electrical attraction to either electrode). As a result, the proteins focus into a sharp stationary band, with each protein located at the point in the pH gradient corresponding to its pI.

[0028] Capillary isoelectric focusing (CIEF) and imaged capillary isoelectric focusing (iCIEF) Capillary isoelectric focusing (CIEF) and imaged capillary isoelectric focusing (iCIEF) are analytical techniques that use individual protein variants primarily based on their isoelectric point (pI)-specific net charge. In CIEF and iCIEF, sample separation is achieved by focusing the protein sample in an amphipathic pH gradient via an applied electric field. The protein sample is premixed with carrier ampholytes, additives, and a pI marker. The protein sample is then separated in a capillary cartridge equipped with electrolytic tanks at both ends. One tank is filled with acid (anolyte) and the other with base (catholyte). The sample mixture is injected to fill the capillary column, and a voltage is applied to the anolyte and catholyte tanks. This creates a pH gradient that separates and focuses proteins (analytes) based on their pI along the capillary.

[0029] In CIEF, the focusing step is followed by a mobilization step, in which the focused analytes move along the capillary to the capillary outlet and pass a detector, such as a UV or fluorescence detector. Mobilization methods include hydrodynamic or pressure mobilization, i.e., mobilization by applying a gas to the capillary, and chemical mobilization, in which the anolyte or catholyte is replaced with a different electrolyte solution of high ionic strength or different pH.

[0030] In contrast to CIEF, which moves the protein sample across the entire detector, in iCIEF, a whole-column detector, such as a UV or fluorescence detector, monitors the entire process in real time across the entire capillary. This allows for close monitoring of the focusing step and the immediate final charge fluctuation profile thereafter. Therefore, the advantages of iCIEF over other techniques include high resolution, efficient run times, and low sample consumption, making it a suitable technique for protein characterization in the biopharmaceutical industry.

[0031] Variability and ampholyte composition The output of a CIEF or iCIEF analysis of a protein can be an electropherogram. In an electropherogram, the x-axis indicates the isoelectric point or recruitment time. The y-axis indicates the detector reading, usually in absorbance units. Peaks in the electropherogram are sometimes called absorbance peaks. Variations in the composition of the ampholytes can cause variations in the output electropherogram.

[0032] An electropherogram of a reference protein produced using a test ampholyte composition may have one or more of the following changes compared to an electropherogram of the same reference protein produced using a reference ampholyte composition: (1) gain of one or more peaks, (2) loss of one or more peaks, (3) a shift in the pI or migration time of one or more peaks, (4) a change in the area under the peak of one or more peaks, and (4) a change in the resolution of one or more peaks.

[0033] Thus, the present disclosure provides a method for determining whether a test ampholyte composition produces an electropherogram of a reference protein similar to an electropherogram of the same reference protein produced by a reference ampholyte. In some embodiments, the method includes: (a) using liquid chromatography-mass spectrometry (LC-MS) to identify at least one marker in at least one test ampholyte composition and in one reference ampholyte composition; and (b) determining the degree of similarity or difference of the at least one marker between the at least one test ampholyte composition and the reference ampholyte composition, wherein the at least one test ampholyte composition has appropriate activity if the at least one marker has low covariance and the level of the at least one marker is different between the at least one test ampholyte composition and the reference ampholyte composition, or the at least one test ampholyte composition has appropriate activity if the at least one marker has high covariance and the level of the at least one marker is similar between the at least one test ampholyte composition and the reference ampholyte composition, thereby identifying a test ampholyte composition with appropriate activity.

[0034] The present disclosure further provides a method for verifying the activity of an ampholyte composition characterized using the methods described herein, comprising generating electropherograms using a reference ampholyte composition and at least one test composition, and comparing the electropherograms to determine whether they are similar.

[0035] In some embodiments, the similarity of at least one test electropherogram and a reference electropherogram is determined by the number, size, resolution, or isoelectric point (pI) of peaks, or a combination thereof.

[0036] In some embodiments, similar electropherograms have a similar or identical number of peaks (e.g., the number of peaks differs by 3, 2, 1, or 0 peaks). As a further example, both the test electropherogram and the reference electropherogram comprise or consist essentially of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 peaks.

[0037] In some embodiments, the similarity of at least one test electropherogram and a reference electropherogram comprises similar resolution.

[0038] As used herein, "resolution" or "R" is a measure of the quality of a separation, e.g., the separation of a pair of absorbance peaks in an electropherogram. A method for calculating the resolution between two peaks is shown in Figures 5A-5C. A resolution of 1.5 (0.15% mutual overlap) is considered the minimum standard for baseline separation and chromatography of two peaks. The higher the resolution, the better the separation of two peaks. Resolution is a combination of two factors: selectivity and efficiency of the separation. Selectivity considers the distance between the maxima of two peaks. The more efficient the separation, the narrower the peaks. Therefore, narrow peaks and large peak-to-peak separations result in large R values. The calculation of resolution uses the difference in migration time or pI of the two peaks (Rt1, Rt2) and the base width of the peaks (W1, W2).

[0039] In some embodiments, the similarity between the test electropherogram and the reference electropherogram comprises a difference in resolution of less than 0.30, less than 0.25, less than 0.20, less than 0.175, less than 0.15, less than 0.13, less than 0.12, less than 0.11, less than 0.10, less than 0.08, less than 0.075, less than 0.07, less than 0.05, less than 0.03, less than 0.02, or less than 0.01. In some embodiments, the similarity between the test electropherogram and the reference electropherogram comprises a difference in resolution of less than 0.075. In some embodiments, the similarity between the test electropherogram and the reference electropherogram comprises a difference in resolution of less than 0.072.

[0040] In some embodiments, the similarity between the test and reference electropherograms includes one or more peaks within regions of the electropherograms that have similar regions. In some embodiments, the difference in the areas of one or more peaks in a particular region of the test and reference electropherograms has a probability value (p-value) of 0.05 or less. Methods for calculating the area under a peak are well known to those skilled in the art and include, for example, fitting a curve equation to the peak and integrating to find the enclosed area. In some embodiments, the particular region of the reference and test electropherograms from which the peak area is calculated can be defined by a particular pI marker contained in the analyte mixture. In some embodiments, the particular region of the reference and test electropherograms from which the peak area is calculated can be defined by the presence of a major peak common to the electropherograms. For example, the electropherograms can be divided into a major peak and an acidic or basic region relative to the major peak.

[0041] Significance can be determined by calculating a p-value or probability value, which is the probability that a null model (e.g., two peaks having the same area by chance) is true. Methods for calculating p-values include Student's t-test, chi-square test, analysis of variance (ANOVA), Pearson correlation coefficient, and Bonferroni-Dan, and are well known to those skilled in the art. In some embodiments, a result is considered significant if the p-value is less than 0.05. In some embodiments, a result is considered significant if the p-value is less than 0.04, less than 0.03, less than 0.02, or less than 0.01.

[0042] In some embodiments, the difference in the area of one or more peaks in a particular region of the test electropherogram and the reference electropherogram has a coefficient of variation (%RSD) of 5% or less. The percent RSD is defined as the ratio of the standard deviation to the mean and indicates the degree of variation relative to the mean of a population (e.g., replicate electropherograms). Methods for calculating the percent RSD will be known to those skilled in the art. In some embodiments, a dataset is reproducible if the %RSD is less than 5%. In some embodiments, a dataset is reproducible if the %RSD is less than 4%, less than 3%, less than 2%, or less than 1%.

[0043] Characterization of ampholyte composition using liquid chromatography-mass spectrometry (LC-MS) Provided herein are methods for characterizing ampholyte compositions using liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the methods include using liquid chromatography-mass spectrometry (LC-MS) to identify at least one marker in at least one test ampholyte composition and in a reference ampholyte composition and determining the degree of similarity or difference of the at least one marker between the at least one test ampholyte composition and the reference ampholyte composition. The at least one marker can be distinguished by its mass-to-charge ratio (m / z) in MS, as discussed below, and the level of the at least one marker is determined by the relative intensity measured by MS for each marker at a given m / z. In some embodiments, the methods include using liquid chromatography-mass spectrometry (LC-MS) to identify multiple markers in the at least one test ampholyte composition and in the reference ampholyte composition and determining the degree of similarity or difference of the multiple markers between the at least one test ampholyte composition and the reference ampholyte composition.

[0044] Liquid chromatography-mass spectrometry (LC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (e.g., high-performance liquid chromatography, or HPLC) with the mass analysis capabilities of mass spectrometry (MS). Liquid chromatography separates mixtures containing multiple components, while mass spectrometry provides the structural identity and levels of individual components with high molecular specificity and detection sensitivity.

[0045] Liquid chromatography and mass spectrometry are described in EP3143392, the contents of which are incorporated herein by reference.

[0046] As used herein, the term "chromatography" refers to a process in which a chemical mixture, including a liquid or gas, is separated into components as a result of differential distribution of chemicals flowing around, over, and / or through a stationary liquid or solid phase. "Liquid chromatography" or "LC" refers to a process in which one or more components of a fluid solution are selectively retarded as the fluid permeates uniformly through a column or capillary passage of finely divided material. Retardation results from the distribution of the mixture's components between one or more stationary phases and the bulk fluid (i.e., the mobile phase) as the fluid moves relative to the stationary phase. Liquid chromatography includes, but is not limited to, reversed-phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC), high-speed turbulent liquid chromatography (HTLC), and ultra-high performance liquid chromatography. "Retention time" refers to the length of time a particular analyte, such as an ampholyte composition component, is retained by the liquid chromatography substrate before elution.

[0047] As used herein, the term "high performance liquid chromatography" or "HPLC" refers to liquid chromatography in which the degree of separation is increased by forcing a mobile phase under pressure through a stationary phase, typically a tightly packed column.

[0048] As used herein, the term "ultra-performance liquid chromatography" or "UPLC" refers to a liquid chromatography method that offers improved speed, sensitivity, and resolution compared to HPLC. Generally, UPLC is applicable to particles less than 2 μm in diameter. Separation and quantitation in UPLC is performed under very high pressure (up to 100 MPa).

[0049] Gas chromatography (GC) refers to a separation technique that uses a gas flowing through a column, such as a glass or metal column, to separate compounds based on their volatility and interaction with a liquid stationary phase. The carrier gas, or mobile phase, is usually an inert gas such as helium or nitrogen.

[0050] Mass spectrometry is performed using a mass spectrometer, which includes an ion source for ionizing the sample and creating charged molecules for further analysis. In various embodiments, the ampholyte composition and its components can be ionized by any method known to those skilled in the art. Ionization sources used in various MS techniques include, but are not limited to, electron ionization, chemical ionization, electrospray ionization (ESI), photoionization, atmospheric pressure chemical ionization (APCT), photoionization, atmospheric pressure photoionization (APPI), fast atom bombardment (FAB) / liquid secondary ionization (LSIMS), matrix-assisted laser desorption / ionization (MALDI), field ionization, field desorption, thermospray / plasma spray ionization, surface-enhanced laser desorption / ionization (SELDI), inductively coupled plasma (ICP), particle beam ionization, and ion mobility separation (IMS). Those skilled in the art will understand that the choice of ionization method can be determined based on the analyte to be measured, the type of sample, the type of detector, the choice of positive mode versus negative mode, and the like.

[0051] As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique for identifying compounds by their mass. MS refers to a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio (m / z). MS techniques generally involve ionizing compounds to form charged species (e.g., ions) and detecting the ions' exact mass divided by their charge (m / z). Compounds can be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. Generally, one or more molecules of interest are ionized, and the ions are then introduced into a mass analysis instrument, where a combination of magnetic and electric fields causes the ions to follow a path in space that depends on their mass ("m") and charge ("z"). See, e.g., U.S. Patent Nos. 6,204,500, 6,107,623, 6,268,144, and 6,124,137.

[0052] MS can generate and detect both positive and negative ions. As used herein, the terms "ionization" or "ionize" refer to the process of producing analyte ions with a net charge equal to one or more electron units. A positive ion is an ion with a net positive charge of one or more electron units. A negative ion is an ion with a net negative charge of one or more electron units. In "electron ionization" or "EI" techniques, a gas- or vapor-phase analyte interacts with a stream of electrons. When the electrons strike the analyte, analyte ions are produced, which can then be subjected to mass spectrometry techniques. EI can be combined with gas chromatography (GC) or liquid chromatography techniques. In "chemical ionization" or "CI," a reagent gas (such as ammonia) is bombarded with electrons, and analyte ions are formed by the interaction of the reagent gas ions with analyte molecules. In "fast atom bombardment" or "FAB," a beam of energetic atoms (often Xe or Ar) strikes a nonvolatile sample, desorbing and ionizing the molecules contained in the sample. Test samples are dissolved in a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2-nitrophenyl octyl ether, sulfolane, diethanolamine, triethanolamine, etc. Selecting an appropriate matrix for a compound or sample is an empirical process.

[0053] As used herein, the term "matrix-assisted laser desorption / ionization" or "MALDI" refers to a method in which a nonvolatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample through various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. In MALDI, the sample is mixed with an energy-absorbing matrix, which facilitates desorption of analyte molecules. MALDI-TOF refers to matrix-assisted laser desorption / ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS). MALDI-TOF is useful for compounds up to approximately 15,000 daltons.

[0054] "Surface-enhanced laser desorption ionization" or "SELDI" refers to another method in which a nonvolatile sample is exposed to laser irradiation, which desorbs and ionizes analytes within the sample through various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. In SELDI, the sample is typically bound to a surface that preferentially retains one or more analytes of interest. As with MALDI, energy-absorbing materials can be used to facilitate ionization in this process.

[0055] "Electrospray ionization" or "ESI" refers to a method in which a solution is passed along a short length of capillary tube, at the end of which a high positive or negative potential is applied. The solution reaching the end of the tube is vaporized (atomized) into a jet or spray of very small droplets of the solution in solvent vapor. This mist of droplets flows through an evaporation chamber, which is slightly heated to prevent condensation and evaporate the solvent. As the droplets become smaller, the electrical surface charge density increases until natural repulsion between like charges releases ions and neutral molecules.

[0056] "Atmospheric pressure chemical ionization" or "APCI" refers to a mass spectrometry method similar to ESI, except that APCI generates ions through ion-molecule reactions occurring within a plasma at atmospheric pressure. The plasma is maintained by an electrical discharge between a spray capillary and a counter electrode. The ions are then typically extracted into a mass spectrometer using a set of differential pump-skimmer stages. A counterflow of dry, preheated N2 gas may be used to improve solvent removal. APCI's gas-phase ionization is more effective than ESI for the analysis of less polar chemical species.

[0057] As used herein, the term "atmospheric pressure photoionization" or "APPI" refers to a form of mass spectrometry in which the mechanism of photoionization of a molecule M is photon absorption and electron ejection to form a molecule M+. Typically, the photon energy is just above the ionization potential, making the molecular ion less susceptible to dissociation.

[0058] As used herein, the term "inductively coupled plasma" or "ICP" refers to a method in which a sample interacts with a partially ionized gas at a high enough temperature to atomize and ionize most elements.

[0059] As used herein, "ion mobility spectrometry," also known as "ion mobility separation" or "IMS," refers to an analytical chemistry method that separates gas-phase ions based on their interaction with a collision gas and their mass. In a first step, an ion mobility spectrometer is used to separate ions according to their mobility through a buffer gas on the millisecond timescale. The separated ions are then, in a second step, introduced into a mass spectrometer, which can determine their mass-to-charge ratio on the microsecond timescale.

[0060] As used herein, the term "field desorption" refers to a method in which a non-volatile test sample is placed on an ionizing surface and a strong electric field is used to generate analyte ions.

[0061] As used herein, the term "desorption" refers to the removal of an analyte from a surface and / or the entry of the analyte into the gas phase.

[0062] After the ampholyte composition or its components are ionized, the ions produced thereby can be analyzed to determine m / z. Suitable analytical devices for determining m / z include quadrupole analyzers, ion trap analyzers, time-of-flight analyzers, Fourier transform ion cyclotron resonance (FTICR) analyzers, and Orbitrap spectrometers. Ions can be detected using one of several detection modes. For example, only selected ions can be detected using selective ion monitoring mode (SIM), or multiple ions can be detected using a scanning mode, such as multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).

[0063] In some embodiments, m / z is determined using a quadrupole analyzer (instrument). In a "quadrupole" or "quadrupole ion trap" instrument, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between the electrodes, the amplitude of the RF signal, and m / z. The voltage and amplitude can be selected so that only ions with a specific m / z travel the length of the quadrupole while all other ions are deflected. Thus, a quadrupole instrument can function as both a "mass filter" and a "mass detector" for ions injected into the instrument. In time-of-flight (ToF), ions are accelerated in a uniform electrostatic field using ground and repeller electrodes. Their kinetic energy is kept constant, and they travel through a field-free ToF tube. Because kinetic energy is constant (KE=1 / 2MV^2), ions with lower m / z will experience faster velocities than ions with higher m / z. "Quadrupole Time-of-Flight" or "QTof" mass spectrometry refers to a type of mass spectrometry that uses a mass spectrometer that combines a quadrupole acting as a collision cell with a time-of-flight analyzer. This allows all ions to be analyzed simultaneously with high resolution and mass accuracy. In an exemplary QTof system, a sample is delivered and ionized by an online liquid chromatography system. The particle beam then travels through an ion guide to the quadrupole before being passed to the ToF analyzer. In some embodiments, the MS technique can use "tandem mass spectrometry" or "MS / MS." In this technique, precursor ions (also called parent ions) generated from a molecule of interest can be filtered in the MS instrument, which then fragments the precursor ions to produce one or more fragment ions (also called daughter ions or product ions) that are subsequently analyzed in a second MS step. By carefully selecting precursor ions, only ions generated by a specific analyte are passed to the fragmentation chamber, where fragment ions are generated by collisions with atoms of an inert gas. Because both precursor and fragment ions are generated in a reproducible manner under a specific set of ionization / fragmentation conditions, the MS / MS technique can provide a very powerful analytical tool.For example, a filtration / fractionation combination can be used to eliminate interfering substances and can be particularly useful in complex samples such as biological samples.

[0064] Mass spectrometers typically provide users with an ion scan or mass spectrum, i.e., the relative abundance of each ion with a specific m / z in a specific range (e.g., 400-1600 m / z). The mass spectrum can be related to the amount of an analyte in a sample, such as a component of an ampholyte composition, by many methods known in the art. For example, if sampling and analysis parameters are carefully controlled, the relative abundance (also called relative intensity) of a given ion can be compared to a table that converts that relative abundance to the absolute amount of the original molecule. Alternatively, molecular standards can be generated using a standard curve created based on the ion signals generated from the sample and those standards. Methods for generating and using such standard curves are well known in the art, and one of skill in the art can select an appropriate internal standard. Many other methods for relating the amount of an ion to the amount of the original molecule will be familiar to those of skill in the art.

[0065] When ions strike the detector, a pulse of electrons is generated and converted into a digital signal. The acquired data is relayed to a computer, which plots the ion counts per unit time. The area under the peak corresponding to a particular ion, or the amplitude of such a peak, is measured, and the area or amplitude correlates with the amount of the target component or marker in the ampholyte composition. In certain embodiments, the area under the curve or the amplitude of the peak of the fragment ion and / or precursor ion is measured to determine the amount of an analyte having a given m / z. As described above, the relative abundance, sometimes referred to as relative intensity, or the response of a particular ion can be converted to the absolute amount of the original analyte using a calibration standard curve based on the peaks of one or more ions of an internal molecular standard. The absolute amount of the ampholyte composition component detected by LC-MS can then be converted to the absolute amount of the component present in the original sample.

[0066] Relative abundance or relative intensity can also be defined as the y-axis of a mass spectrum. In some embodiments, the amount of a marker can be quantified relative to the amount of the most abundant ion (base peak) in the mass spectrum. In some embodiments, the amount of a marker can be quantified using a response factor or a standard curve. In some embodiments, quantifying the amount of a marker includes (1) including an internal standard, e.g., a reference ampholyte and / or a test ampholyte composition, in the sample, and (2) normalizing the marker to the internal standard in the sample. In some embodiments, quantifying the amount of a marker further includes (3) normalizing the marker to the maximum corrected amount of the marker measured across multiple ampholyte compositions. For example, the marker can be normalized to the maximum corrected response (also known as the relative intensity) of the marker measured from multiple ampholyte lots from the same manufacturer. The internal standard can include a standard that elutes mid-run and exhibits high ionization efficiency. 13 Includes, but is not limited to, C6-carbamazepine.

[0067] In some embodiments, the response of an internal standard can be used to normalize the response values for each m / z to account for instrument drift.

[0068] In some embodiments, changes in retention time (RT), collision cross section (CCS), or both, and the m / z of the running internal standard can be used to set tolerances on the parameters of marker assignment.

[0069] In some embodiments, the relative intensity of a marker or component of an ampholyte composition is normalized, for example, to the maximum relative intensity of that marker measured across multiple ampholyte compositions.

[0070] As used herein, "accurate mass," sometimes referred to as "measured accurate mass," is the experimentally determined mass from MS that allows for the determination of the elemental composition of an analyte, such as an ampholyte composition component. "Accurate Mass / Retention Time" or "AMRT" refers to the combination of (1) the retention time of a component of an ampholyte composition during liquid chromatography and (2) the exact mass of the component as measured by mass spectrometry.

[0071] As used herein, "collision cross section" or CCS data can be obtained through ion mobility experiments. This is the effective region for interactions between individual ions and neutral gas molecules and is related to the ion's properties, such as chemical structure and dimensions. It can be derived using methods such as drift tube ion mobility measurements or traveling wave IMS. For example, multiple measurements are performed at various electric fields to calculate the CCS value.

[0072] In some embodiments, the LC-MS comprises ultra-performance liquid chromatography tandem ion mobility quadrupole time-of-flight mass spectrometry (UPLC-IMS-Q-Tof_MS).

[0073] Suitable LC-MS instruments and systems include, but are not limited to, the BioAccord LC-MS System for Biopharmaceuticals (Waters), the Waters Acquity UPLC H-Class coupled with a Waters Vion IMS-Q-ToF-MS, the Agilent Ultivo LC / MS system, and the Orbitrap LC-MS system (ThermoFisher).

[0074] Identification of markers of ampholyte composition The present disclosure provides a method for identifying at least one marker in an ampholyte composition, the method comprising: (i) determining accurate mass / retention time (AMRT) or collision cross section measurements of a plurality of components of at least one test ampholyte composition and a reference ampholyte composition; (ii) using an S-plot to plot the covariance of the AMRT measurements of the plurality of components from the at least one test ampholyte composition and from the reference ampholyte composition; and (iii) selecting at least one component that differs between the at least one test ampholyte composition and the reference ampholyte composition, or selecting at least one component that is similar between the at least one test ampholyte composition and the reference ampholyte composition, thereby identifying at least one marker for characterizing the suitability of the at least one test ampholyte composition.

[0075] The similarities and differences between the AMRT measurements of ampholyte composition components can be determined by any statistical method known in the art. One approach involves calculating and plotting the covariance of AMRT pairs between components of at least one test ampholyte composition and a reference composition. As used herein, covariance refers to the joint variability of two random variables. If the larger values of one variable correspond primarily to the larger values of the other variable, and the same is true for the smaller values (i.e., if the variables tend to behave similarly), the covariance is positive. Conversely, if the larger values of one variable correspond primarily to the smaller values of the other variable (i.e., if the variables tend to behave in opposite ways), the covariance is negative.

[0076] Covariance can be calculated using methods such as principal component analysis (PCA), orthogonal projections onto latent structures - discriminant analysis (OPLS-DA), Waters S-plots, etc., which will be well known to those skilled in the art.

[0077] "Principal Component Analysis" or "PCA" is a statistical procedure that uses an orthogonal transformation to convert a set of potentially correlated variables into a set of linearly uncorrelated variable values, called principal components. This transformation is defined so that the first principal component has the greatest possible variance (i.e., explains as much of the variation in the data as possible), and each subsequent component has the greatest possible variance under the constraint that it is orthogonal to the previous component. Thus, PCA reduces large sets of multivariate data to uncorrelated variables, called principal components. PCA is limited in that information about individual sample groups cannot be ascertained from the plot.

[0078] In some embodiments, the data can be further mined using orthogonal projection onto latent structures - discriminant analysis (OPLS-DA), a statistical analysis that allows the identification of specific features (e.g., ampholyte species) that contribute to the overall differences observed between the two groups.

[0079] In some embodiments, PCA and, optionally, OPLS-DA data can be viewed as an S-plot. An S-plot is a statistical method for plotting the dissimilarity between two groups of values. Thus, an S-plot compares two samples (e.g., a minimum of n=3 measurements). For example, AMRT pairs and / or CCS measurements of components from a reference and test ampholyte composition can be plotted using an S-plot. In an S-plot, covariance or magnitude of change is plotted on the x-axis, and correlation, i.e., consistency of change, is plotted on the y-axis. The farther a marker is placed from the origin of the x-axis, the greater its contribution to variance between groups, and markers farther along the y-axis represent higher confidence in the analytical results. In an exemplary S-plot, the covariance of AMRT pairs from at least one test ampholyte composition and a reference ampholyte is plotted. Every point on the S-plot is a specific m / z with associated RT and CCS values. Markers are plotted by consistency across replicates and magnitude of change. The further away from the origin along the x-axis, the greater the difference in intensity (or relative concentration) between samples. Differences between groups can be due to markers that are only present in one group or that vary greatly in intensity between groups. The further away from the origin on the y-axis, the more confidence there is in the analytical results across triplicates. An S-plot can be used to identify the features that differ most between lots, and these markers can be used to generate a screening library to characterize ampholyte lots.

[0080] In some embodiments, a method for identifying at least one marker for determining the suitability of an ampholyte composition includes: (i) determining accurate mass / retention time (AMRT) measurements of a plurality of components of at least one test ampholyte composition and a reference ampholyte composition; (ii) using an S-plot to plot the covariance of the AMRT measurements of the plurality of components from the at least one test ampholyte composition and the reference ampholyte composition; and (iii) selecting at least one component that differs between the at least one test ampholyte composition and the reference ampholyte composition, wherein the difference comprises a covariance that is less than zero in the S-plot, thereby identifying at least one marker for characterizing the suitability of the at least one test ampholyte composition. If the covariance is less than zero in the S-plot, the paired AMRT values are negatively correlated between the reference and test ampholyte compositions. Thus, at least one marker is selected based on a difference between the reference ampholyte composition and the test ampholyte composition, and the difference in the presence and / or level of the at least one marker between the reference and test ampholyte compositions determines the suitability of the test ampholyte composition.

[0081] In some embodiments, e.g., embodiments in which at least one marker is a marker of difference between a reference and a test ampholyte composition, the at least one component selected as a marker comprises a covariance in the S-plot that is less than the covariance of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the components in the S-plot.

[0082] In some embodiments, the difference in the level of at least one marker comprises a difference in level of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% relative to the normalized level of the at least one marker. For example, a reference ampholyte composition comprises a normalized level of at least one marker that is 100%, and a test ampholyte composition comprises a normalized level of at least one marker that is 30%. Markers can be characterized by their mass-to-charge ratio (m / z) and the level of the marker as determined by their relative intensity normalized to the maximum relative intensity as described herein.

[0083] In some embodiments, a method for identifying at least one marker for determining the suitability of an ampholyte composition includes: (i) determining accurate mass / retention time (AMRT) measurements of multiple components of at least one test ampholyte composition and a reference ampholyte composition; (ii) using an S-plot to plot the covariance of the AMRT measurements of multiple components from the at least one test ampholyte composition and the reference ampholyte composition; and (iii) selecting at least one component that is similar between the at least one test ampholyte composition and the reference ampholyte composition, where the similarity comprises a covariance that is greater than zero in the S-plot, thereby identifying at least one marker for characterizing the suitability of the at least one test ampholyte composition. If the covariance is greater than zero in the S-plot, the paired AMRT values are positively correlated between the reference and test ampholyte compositions. Thus, at least one marker is selected based on the similarity between the reference ampholyte composition and the test ampholyte composition, and the similarity in the presence and / or level of the at least one marker between the reference and test ampholyte compositions determines the suitability of the test ampholyte composition.

[0084] In some embodiments, e.g., embodiments in which at least one marker is a marker of similarity between the reference and test ampholyte compositions, the at least one component selected as a marker comprises a covariance in the S-plot that is greater than the covariance of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the components in the S-plot.

[0085] In some embodiments, at least one test ampholyte composition has appropriate activity if the level of at least one marker in at least one test ampholyte composition and the reference ampholyte composition are similar. In some embodiments, similarity in the level of at least one marker includes levels that are at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% similar compared to the normalized level of the at least one marker. For example, the reference ampholyte composition contains a normalized level of at least one marker that is 100%, and the test ampholyte composition contains a normalized level of at least one marker that is at least 90%. As a further example, the reference ampholyte composition contains a normalized level of at least one marker that is 100%, and the test ampholyte composition contains a normalized level of at least one marker that is at least 80%. Markers can be characterized by mass-to-charge ratio (m / z) and the level of the marker determined by relative intensity normalized to the maximum relative intensity as described herein.

[0086] The present disclosure provides a method for determining the degree of similarity or difference of at least one marker between at least one test electrolyte composition and a reference ampholyte composition. In some embodiments, the method includes (i) determining LC-MS mass spectra of at least one marker in at least one test ampholyte composition and in the reference ampholyte composition, (ii) determining the relative intensity of the at least one marker in the mass spectra of the at least one test ampholyte composition and the reference ampholyte composition, (iv) normalizing the relative intensity of the base peak of the at least one marker to the maximum relative intensity of the base peak measured from the at least one test ampholyte composition or the reference ampholyte composition, and (iv) comparing the relative intensities of the normalized relative intensities of the base peak of the at least one marker in the at least one test ampholyte composition and the reference ampholyte composition.

[0087] In some embodiments, the at least one marker comprises a plurality of markers, ie, at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, or 100 markers having different m / z.

[0088] In some embodiments, the at least one marker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 markers with different m / z.

[0089] In some embodiments, the at least one marker comprises 16 markers with different m / z, ie, the 16 markers comprise markers at m / z 280, m / z 319, m / z 329, m / z 347, m / z 373, m / z 375, m / z 376, m / z 431, m / z 504, m / z 506, m / z 508, m / z 520, m / z 534, m / z 562, m / z 906, and m / z 980.

[0090] Suitable multiple markers of different m / z that can be used to characterize a test ampholyte composition can be determined de novo for a reference ampholyte composition using the methods described herein, for example, by characterizing the reference and test ampholyte compositions via LC-MS, plotting AMRT versus S-plots, and selecting at least one or more markers based on covariance.

[0091] Alternatively, or in addition, a test ampholyte composition can be characterized using the methods described herein through comparison to a predetermined library of markers previously determined using the methods described herein. For example, the relative intensity of at least one or more markers of a test ampholyte composition can be determined via LC-MS and compared to a predetermined reference or standard library of markers.

[0092] The present description sets forth numerous example configurations, methods, parameters, etc. However, it should be recognized that such descriptions are not intended to limit the scope of the present disclosure, but rather are provided as descriptions of example embodiments. [Example]

[0093] Example 1: Imaged Capillary Isoelectric Focusing (iCIEF) Protocol for Determining Protein Charge Variant Levels and / or Determining Protein Product Identity iCIEF is used for laboratory testing of drug substances, formulated drug substances, drug products, and in-process materials for which written specifications have been established. When used for release testing against specifications, iCIEF standard operating procedures (SOPs) must be followed and deviations addressed. Below is an example iCIEF SOP protocol where results are affected by lot-to-lot variability of ampholyte lots used in the SOP protocol.

[0094] definition iCIEF - Imaged capillary isoelectric focusing. pI - isoelectric point, the pH of a molecule at which the net charge is zero. Region 1 (acidic) - A group of peaks that are relatively acidic compared to the largest peak in the iCIEF electropherogram. Region 2 (neutral) - major peak corresponding to the largest protein peak / peaks. Region 3 (basic) - A relatively basic group of peaks compared to the largest peak in the iCIEF electropherogram. Zwitterions - Compounds containing both positive and negative charges that behave as zwitterions at and near their pI values. Ampholytes assist in the formation of pH gradients during focusing. Transfer time measurement - an experiment performed to ensure proper sample injection. The measurement is performed when starting up the iCE3 instrument or when installing a new cartridge. Premix mode - Protein samples are premixed with the carrier Pharmalyte, additives, and pI markers.

[0095] material Safe-Lock Eppendorf Tubes, 1.5 mL (VWR, cat. #21008-959) or similar tubes of the same construction cIEF cartridge FC coating (ProteinSimple, PN101701) 10mL Amber Vials, 22x75, with Caps (Thin Septa) (ProteinSimple, PN045-139) 300 μL polypropylene vial with molded insert (ProteinSimple, PN045-133) Septum Pack iCE3 (300 μL cap) (ProteinSimple, PN045-134) Electrolyte pipette (ProteinSimple, PN101788) or a similar pipette with the same structure Microinjection transfer capillary, coated (ProteinSimple, PN102694) 5mL microtubes, natural (Argos Technologies, Cat. No. T2076), or similar tubes of the same construction Amicon Ultra-0.5 centrifugal filter units with Ultracel-10 membrane (Millipore, catalog number UFC501096)

[0096] chemical products Water purified with a Milli-Q system or purchased water such as HPLC Grade Water (VWR, cat. #JT4218-3), or water for injection (WFI), (APP Pharmaceuticals, cat. #918510) Appropriate antibody reference standard (current standard lot) ·Urea (Sigma-Aldrich, BioXtracat#U0631) · 3-10 Pharmalyte (VWR Catalog No. 17-0456-01 or Sigma Catalog No. P1522-25ML) Store at 2-8°C. 0.5% methylcellulose (MC) solution (ProteinSimple, PN102505) 1% methylcellulose (MC) solution (ProteinSimple, PN101876) Electrolyte Kit (ProteinSimple, PN102506): ○100mL0.08MH3PO4 ○100mL 0.1M NaOH pI marker pI5.12 (ProteinSimple, PN102224) pI marker pI 9.50 (ProteinSimple, PN101996)

[0097] Device Microcentrifuge for test preparations ProteinSimple iCE3 Charge Variant Analyzer VWR single channel pipettes (VWR#89130-554, 556, 560, 562, 566) or similar range pipettes Sterile pipette tips (VWR) or tips that match the pipette you are using ·Positional Volumetric Pipettes (Rainin#MR-10, MR-100, MR-250, MR-1000) Rainin positive pipette tips (Rainin #C-10, C-100, C-250, C-1000) Eppendorf Repeater Plus Pipettor (VWR, Catalog No. 21516-002) Eppendorf Combitips pipette tips (VWR, catalog number 21516-138) ·Analysis balance

[0098] Reagent solutions The amount of reagent solution can be adjusted as needed.

[0099] 8M Urea Solution: To prepare an 8M urea solution, weigh 2.4 g of urea into a 5 mL conical tube. Slowly add water to approximately the 4 mL mark. Stir the mixture until the urea is dissolved. Bring the solution to a final volume of 5 mL with water. Mix well. Prepare fresh daily.

[0100] Master Mix: The following is a single preparation that should be scaled as needed. For the general iCIEF method, a pI 5.12 marker and a pI 9.50 marker are recommended as acidic and basic markers. The master mix should be mixed well and prepared fresh daily.

[0101] Table 1: Master mix TIFF2025114703000002.tif50128

[0102] procedure You can use the standard boot procedure to start iCE3.

[0103] Reference Standard Preparation: A current reference standard can be used to demonstrate system suitability. Dilute the appropriate reference sample to 2 mg / mL with water. Mix the standard by gently inverting and spin the tube in a microcentrifuge for approximately 15 seconds.

[0104] In a separate tube, combine 40 μL of a 2 mg / mL reference standard or test article preparation with 160 μL of master mix. At least two vials should be prepared for the required system suitability injection. This preparation can be scaled accordingly. Mix by gently inverting and vortexing for approximately 15 seconds to push the contents to the bottom of the tube.

[0105] In-process sample preparation: Dilute the in-process sample to 2 mg / mL using water. Mix by gently inverting. Assemble the centrifugal filter device by placing the filter in a clean microcentrifuge tube. Pre-wet the filter membrane by pipetting 400 μL of water onto the filter, ensuring the entire membrane is wet. Remove the water from the filter by pipetting and discard the water in a waste container. Load 200 μL of the diluted 2 mg / mL in-process sample onto the pre-wet filter. Place the microcentrifuge tube over the attached filter and close the centrifugal filter device. Centrifuge the device for 15 minutes at 14,000 relative centrifugal force (rcf).

[0106] Remove the device from the centrifuge. Remove the filter from the device and invert it into a clean microcentrifuge tube. Discard the microcentrifuge tube containing the removed buffer. If you place the filter in the clean microcentrifuge tube, you will not be able to put the cap back on. Place the uncapped device with the inverted filter in the centrifuge and spin at 1,000 rcf for 2 minutes. After subsequent centrifugation, the concentrated protein will be in the microcentrifuge tube. Discard the filter.

[0107] Reconstitute the concentrated protein in 180 μL of water. Mix by gently inverting and spin for approximately 15 seconds to push the contents to the bottom of the tube.

[0108] Mix 40 µL of the filtered sample with 160 µL of the master mix. Gently invert to mix and spin for approximately 15 seconds to push the contents to the bottom of the tube.

[0109] Preparation of Drug Substance, Formulated Drug Substance and / or Drug Product: If the sample concentration is less than 20 mg / mL, the buffer exchange procedure above for in-process samples should be followed in lieu of the protocol below for Preparation of Drug Substance, Formulated Drug Substance and / or Drug Product.

[0110] If the drug substance, formulated drug substance, or drug product is an antibody, do not vortex at any time during preparation.

[0111] Dilute the test article (drug substance, formulated drug substance, or drug product) to 2 mg / mL with water. Mix by gentle inversion and spin the tube in a microcentrifuge for approximately 15 seconds. In a separate tube, combine 40 μL of the 2 mg / mL test article preparation with 160 μL of master mix. Mix by gentle inversion and spin for approximately 15 seconds to force the contents to the bottom of the tube.

[0112] Prepare blank: Mix 40 μL of water with 160 μL of master mix. Gently invert to mix and spin for approximately 15 seconds to push the contents to the bottom of the tube.

[0113] Sample loading using the iCE3 instrument: Use the iCE3 premix mode. Transfer 150 μL of the supernatant from the samples (reference standard / reference solution, test article, blank preparation) prepared as described above into a polypropylene vial with a 300 μL molded insert. Ensure there are no air bubbles at the bottom of the vial.

[0114] Place the sample vial into the Alcott 720 autosampler. Fill a 10 mL amber vial with approximately 8 mL of 0.5% methylcellulose (MC) and place it in the "D" position of the autosampler. Use fresh MC solution daily to prevent microbial growth. Insert the water line into a new bottle of Milli-Q water and place it on top of the instrument. Place the waste line in the designated waste container.

[0115] Batch Setup and Start (iCE3): Open the CFR software. Click on "Batch / Data" and select "Deploy." The "Batch File Control" window will appear. Create a batch file using one of the following options: (1) To create a new batch file, enter the batch file name, select the folder to save the batch in, select the number of injection runs, and click New File. (2) To start a new batch file using a previously saved batch file as a template, locate the desired file in the left pane of the Batch File Control window. Click Open File. Click the Save As button, enter a new file name, and click OK.

[0116] In a batch, the first injection should be a blank. The test article should be bracketed with at least five injections of the appropriate product reference standard. It is recommended to make three reference standard injections before the test article injection, followed by two reference standard injections, for a total of five reference standard injections.

[0117] Occasionally, injections may be missed or random spikes may occur that interfere with the protein profile, resulting in atypical electropherograms (in terms of intensity, number, and pattern of peaks). Re-injections of blanks, reference standards, and test articles can be performed within 24 hours of the initial injection, using at least five reference standard injections as a bracket, provided that the reference standards used are appropriate for the sample injections involved.

[0118] Set up batch focus time settings: Enter a unique and descriptive file name for each injection. For Sample ID, enter the reference standard / reference solution designation, LIMS number, or other sample identifier. Number vials sequentially. For Focus Time 1 (prefocus), use the following settings: 1 minute at 1500V. For Focus Time 2 (focus), use the following settings: 7 minutes at 3000V. Rinse time is 90 seconds, and transfer time delay is 0.00 minutes.

[0119] Set the autosampler parameters. Temperature Control: Yes; set to 10°C. · Buffer injection time: Default value. Sample injection time: Default value. Loading time: 6 seconds. Vial type: 2mL, 300μL insert. Needle depth: 48mm.

[0120] Sample and buffer injection times are determined during transfer time measurements (startup / cartridge installation system check) and are based on the length of time it takes for a plateau to appear. We recommend at least 10 seconds longer than the plateau time.

[0121] Under sample conditions, enter the following information: Carrier ampholyte: 4% 3-10 Pharmalite Additives: 2M urea, 0.35%MC Low pI marker: pI value of the acidic marker in the master mix (5.12) High pI marker: pI value of basic marker in master mix (9.50) ·Concentration [μg / μL]: 0.40 -Select sample type (optional)

[0122] Regarding concentrations, note that the concentration of the blank is 0. The final concentration of the sample in process may vary depending on the dilution factor used.

[0123] Data Conversion Data conversion can be performed using iCECFR software. The CFR software can be used to generate electropherograms and set pI marks for blank, reference standard, and test sample electropherograms. The CFR software can also be used to convert data into other formats, such as Empower, for further analysis.

[0124] System Compatibility Both acidic (low) and basic (high) pI markers should be present in the electropherograms of the blank, reference standard, and test article injections. Duplicate reference standard injections should be similar in profile (i.e., in terms of intensity, number, and pattern of peaks). Region 1 and Region 2 of at least five replicate reference standard injections should each have an average % area result that meets the product specification of %RSD ≤ 5%.

[0125] Assay Validation The test article profile should be qualitatively similar in intensity, number, and pattern of peaks to the example electropherogram from the product-specific job aid. There should be no peaks in the blank injection protein region of interest to be integrated into the appropriate product-specific job aid.

[0126] Protein charge variant analysis Results are reported to one decimal point. For reference standards, the average replicate % area in Regions 1, 2, and 3 from all injections is reported. The %RSD between all replicate injections in Regions 1, 2, and 3, and the average pI value for Region 2 (for information only) are also reported. For test articles (sometimes called test samples), data is reported as specified in the appropriate product specification or protocol. If no protocol is available, the % area in Regions 1, 2, and 3 is reported. The average pI value for Region 2 is reported for informational purposes only.

[0127] Example 2: Ampholyte lot variation causes differences in electropherograms Ampholytes are key reagents in the iCIEF assay. However, even when analyzing the same reference protein, different lots of ampholytes can show different electropherograms, leading to quality control test failures (compare Figure 1A and 1B).

[0128] Reference Protein 1 was analyzed using iCIEF with Ampholyte Lot 1 (the current standard) and various test ampholyte lots, as described above in Example 1. Table 1 below summarizes the GE ampholyte lots tested during this analysis.

[0129] Table 1: GE Healthcare Pharmalite pH 3-10 lots TIFF2025114703000003.tif44128

[0130] iCIEF electropherograms of reference protein 1, produced with different ampholyte lots but under otherwise identical conditions, showed substantial differences in the profile (e.g., compare Figure 1A with Figure 1B, compare electropherograms in Figure 4A, or Figure 10B).

[0131] Varying drug concentrations in iCIEF analysis showed that as little as a 2% change in ampholyte concentration could cause changes in the electropherogram. For example, changing the ampholyte concentration from 4% to 6% resulted in a 1% decrease in the area of region 1 of reference protein 1 (Figure 3).

[0132] Differences in ampholyte lots also affected the peak resolution of iCIEF electropherograms. As shown in Figures 5A-5C, the resolution of each ampholyte was calculated for pIs between 7.0 and 7.05. As shown in Figures 4A-4B, different ampholyte lots resulted in peak resolutions that varied by 7.0-7.05.

[0133] Example 3: Characterization of Ampholyte Lots by LC-MS and Creation of a Marker Library Traditionally, ampholyte lots have been evaluated indirectly by assessing the performance of an iCIEF assay. The present inventors have developed a method for direct ampholyte lot characterization. These methods analyze the ampholyte itself using LC-MS, rather than analyzing the ability of the ampholyte lot to generate an electropherogram of a known protein reference. This method characterizes the chemical composition of the ampholyte itself by UPLC IMS-Q-ToF-MS and correlates it with the performance of an iCIEF assay using a known reference protein.

[0134] GE ampholyte lot 1 was used to establish the standard operating procedure (SOP) criteria; however, replacement lots were required. Lot-to-lot variability has been observed across GE ampholyte lots, and these differences resulted in different electropherograms during repeated analyses of the same sample, leading to QC test failures. This is seen in Example 2.

[0135] Therefore, we developed a characterization method for ampholytes using UPLC IMS-Q-ToF-MS to evaluate candidate lots. The goal was to chemically characterize ampholyte lots and monitor the components that change between them. This provides a method for characterizing future GE candidate lots of ampholytes and serves as a workflow that can be applied to other vendors. Even without the vendor-provided ampholyte composition, this method allows us to compare candidate lots with current lots using multivariate statistical analysis and evaluate differences.

[0136] As an added benefit, no protein reference standards are consumed using these methods, resulting in a 70:1 sample savings or reduction in sample consumed.

[0137] An overview of the ampholyte testing method is shown in Figure 7. Briefly, ampholyte lots are analyzed neat in triplicate. Ampholyte lots are separated using a Waters VION IMS QTof ion mobility quadrupole time-of-flight mass spectrometry (LC-IMS-Q-TOF-MS) with a C4 column and a full scan to identify components of the ampholyte lot at mass-to-charge ratios (m / z) between 50 and 2000, although this range may vary. Components were assigned within acceptable ranges of ion mobility spectrometry (IMS), m / z, and retention time (RT). Differences between components identified by LC-MS were analyzed by multivariate analysis and plotted using a Waters S-plot. Multivariate analysis identified differences between ampholyte lots used to create a component library that can be used to assess lot-to-lot differences and that will perform similarly to standard ampholyte lots in iCIEF assays using reference proteins.

[0138] FIG. 8A shows the ampholyte lots analyzed by LC-MS to generate the component library.

[0139] Figure 8B shows an exemplary Waters S-plot of ampholyte LC-MS data. The Waters S-plot shows the dissimilarity of accurate mass (AMRT) between two lots of ampholytes. AMRT pairs are plotted by covariance. The magnitude of variation is shown on the x-axis, and the correlation, or consistency of variation, is shown on the y-axis. That is, the further along the x-axis a marker component is located, the greater the contribution of that marker to the variance between ampholyte lots and the greater the difference in marker concentration between lots. The further along the y-axis a marker component is located, the greater the consistency of results between replicates. Differences between ampholytes can be attributed to components present or absent in one or more ampholyte lots and / or variations in the concentration of select markers between lots.

[0140] UPLC IMS-Q-ToF-MS Procedure: Separations were performed on a Waters Acquity UPLC H-Class system equipped with a degasser, chiller, quaternary pump, and autosampler. A Waters BEHTMC4 2.1 x 100 mm analytical column with 1.7 μm particle size was operated at a flow rate of 0.2 mL / min. 10 μL aliquots were used for sample injection. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient began with a 5-minute hold at 95% A and 5% B, and increased linearly to 95% B after 35 minutes. This condition was maintained for 5 minutes, followed by a return to 95% A in 50 minutes. The column was held at this condition for an additional 10 minutes for column re-equilibration. The total run time was 60 minutes.

[0141] A Waters Vion IMS-Q-ToF-MS was used under positive electrospray ionization (+ESI) with accurate mass screening in MSe data mode. Mass spectrometer settings were capillary voltage (2.00 kV), source temperature (150 °C), desolvation temperature (200 °C), cone gas (25 L / h), and desolvation gas (800 L / h). Data collection and analysis were performed using UNIFI™ software version 1.9.4.0.

[0142] Five ampholyte lots (-1, -2, -3, -4, and -5) were evaluated, and 500 μL of each lot was added at 1 μg mL 13 The samples were spiked with 25 μL of C6-carbamazepine (selected as the internal standard due to its intermediate elution and high ionization efficiency). The response of the internal standard was used to normalize the response values of each m / z marker to account for instrument drift. Additionally, the retention time (RT), collision cross section (CCS), and change in m / z of the internal standard during the run were used to set tolerances for the marker assignment parameters. Each ampholyte sample was injected neat from the same vial (n=3). A starting mobile phase composition blank was also analyzed between each lot.

[0143] Data processing began with filtering and statistical analysis. Principal component analysis (PCA) allows for the reduction of large sets of multivariate data into uncorrelated variables called principal components. While PCA has the limitation of not being able to ascertain information about individual sample groups from the plot, data can be further mined using orthogonal projections to latent structure - discriminant analysis (OPLS-DA). This statistical analysis allows for the identification of specific features (i.e., ampholyte species) that contribute to the overall differences observed between the two groups, which can be displayed as an S-plot. The S-plot compares two samples (n = 3 measurements, minimum). Every point on the S-plot represents a specific m / z and has an associated RT and CCS value. Markers are plotted according to their consistency across replicates and magnitude of change. The further away from the origin along the x-axis, the greater the difference in intensity (or relative concentration) between samples. Differences between groups can be attributed to markers present only in one group or markers with large variations in intensity between groups. The further away from the origin on the y-axis, the greater the confidence in the analytical results across triplicates. S-plots were used to identify the most variable features between lots, and these markers were added to a screening library. Selected markers had to be present in all three measurements of the ampholyte sample and not present in the blank. This library was then used to assess the presence or absence of specific markers for each lot, and their presence at specific intensities. Markers suspected of significantly affecting assay performance were further investigated by structural elucidation and database searches for identification.

[0144] result: Figures 10B-10C show the variation in iCIEF profiles generated using lots 1, 3, 4, and 5. iCIEF testing revealed that lot 5 performed most differently compared to lot 1 (Figures 10B-10C). In Figure 10B, the electropherograms grow increasingly different from lot 1, moving downward from lot 1. The electropherogram generated using lot 2 (Figure 10C) was most similar to that generated by lot 1. From the marker library in Figure 10A, lot 2 was predicted to be most similar in ampholyte components to lot 1, the current standard ampholyte lot. As can be seen in Figure 10C, when lot 2 was used to generate an iCIEF profile for Reference Protein 1, this profile was more similar to the lot 1 Reference Protein 1 profile than any of the iCIEF profiles generated by lots 3, 4, or 5 (Figure 10B). Therefore, the ampholyte component library can be used to directly identify ampholyte lots that produce iCIEF profiles similar to the standard lot, lot 1.

[0145] Figures 9A-9C show Waters S-plots comparing three different ampholyte lots with Lot 1, the current standard lot. The 1 vs. 5 S-plot showed that Lot 1 had many features that were absent or of lower intensity in Lot 5 (Figure 9C). The most varied markers were added to the library used to characterize the other ampholyte lots (see Figure 10A). The 1 vs. 2 S-plot in Figure 9A showed agreement between the two lots. This finding was supported by the iCIEF results, which showed that of all GE lots tested, Lot 2 showed results most similar to Lot 1. No markers were selected from this S-plot.

[0146] The boxed dots in Figure 10C indicate components selected for the library of components used to characterize the ampholyte lots. Markers identified from the S-plots were manually evaluated to confirm that they were present in all three runs of the detected lot, were specific to the ampholyte, and were absent from the blank (Figure 11B).

[0147] Additionally, extracted ion chromatograms (XICs) were monitored to confirm the presence of Gaussian peaks (Figure 11A). All markers meeting the criteria were added to a screening library. This library was then used to evaluate which markers were present in which lots and at what intensities. A heat map of the markers of interest was created, and the internal standard-normalized response of each lot was divided by the maximum normalized response across the five lots and multiplied by 100 to obtain a percentage (Figure 10A). The trends observed in the heat map were supported by the results of the iCIEF test. The greater the difference detected between markers from lot 1 and other lots, the greater the electropherogram deviated from the SOP. Markers suspected of having a significant impact on assay performance were further investigated by elucidating their structures and performing database searches for identification using the UNIFI Scientific Information System based on elemental composition calculations, structural database searches, and fragment matching of the high-collision energy data (see Figure 12 for structures and Figure 11A for an example chromatogram used for identification) (Figure 12).

[0148] Conclusion: Characterization of lot-to-lot variability of ampholytes using UPLC IMS-Q-ToF-MS successfully predicted lot performance during iCIEF testing. This method improves the efficiency of selection of new ampholyte lots, reduces the volume of iCIEF testing, and conserves reference standard proteins. This same workflow can work for multiple materials and vendors if future lot-to-lot variability is observed.

Claims

1. 1. A method for identifying a test ampholyte composition having suitable activity, comprising: a. identifying at least one marker in at least one test ampholyte composition and one reference ampholyte composition using liquid chromatography-mass spectrometry (LC-MS); b. determining the degree of similarity or difference of said at least one marker between said at least one test ampholyte composition and said reference ampholyte composition, If the at least one marker has low covariance and the level of the at least one marker differs between the at least one test ampholyte composition and the reference ampholyte composition, then the at least one test ampholyte composition has adequate activity or Alternatively, if the at least one marker has a high covariance and the level of the at least one marker is similar between the at least one test ampholyte composition and the reference ampholyte composition, the at least one test ampholyte composition has suitable activity. said determining; Including, thereby identifying test ampholyte compositions with suitable activity; The method.

2. Step (a) i. determining accurate mass / retention time (AMRT) and / or collision cross section measurements for a plurality of components of the at least one test ampholyte composition and the reference ampholyte composition; ii. plotting the covariance of the AMRT and / or collision cross section measurements of the plurality of components from the at least one test ampholyte composition and from the reference ampholyte composition using an S-plot; iii. Selecting at least one component that differs between the at least one test ampholyte composition and the reference ampholyte composition, wherein the difference comprises a covariance that is non-zero in the S-plot; Including, thereby identifying at least one marker for characterizing the suitability of said at least one test ampholyte composition; The method of claim 1.

3. 3. The method of claim 2, wherein the at least one component comprises a covariance in the S-plot that is less than the covariance of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the components in the S-plot.

4. 4. The method of claim 2 or 3, wherein the at least one test ampholyte composition has a suitable activity if the levels of the at least one marker in the at least one test ampholyte composition and the reference ampholyte composition differ.

5. 5. The method of claim 4, wherein the difference in the level of the at least one marker comprises a difference in level of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% relative to the normalized level of the at least one marker.

6. Step (a) i. determining accurate mass / retention time (AMRT) or collision cross section measurements for a plurality of components of the at least one test ampholyte composition and the reference ampholyte composition; ii. plotting the covariance of the AMRT or collision cross section measurements of the plurality of components from the at least one test ampholyte composition and from the reference ampholyte composition using an S-plot; iii. Selecting at least one component that is similar between the at least one test ampholyte composition and the reference ampholyte composition, wherein the similarity comprises a covariance that is non-zero in the S-plot; Including, thereby identifying at least one marker for characterizing the suitability of said at least one test ampholyte composition; The method of claim 1.

7. 7. The method of claim 6, wherein the at least one component comprises a covariance in the S-plot that is greater than the covariance of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the components in the S-plot.

8. 8. The method of claim 6 or 7, wherein the at least one test ampholyte composition has suitable activity if the levels of the at least one marker in the at least one test ampholyte composition and the reference ampholyte composition are similar.

9. 9. The method of claim 8, wherein the similarity in the level of the at least one marker comprises levels that are at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% similar compared to the normalized level of the at least one marker.

10. The method of any one of claims 1 to 9, wherein the at least one marker is identified by its mass-to-charge ratio (m / z).

11. 11. The method of claim 10, wherein the level of the at least one marker is characterized by the relative intensity of m / z in a mass spectrum.

12. Step (b) i. determining an LC-MS mass spectrum of the at least one marker in the at least one test ampholyte composition and in the reference ampholyte composition; ii. determining the relative intensities of the base peaks of the at least one marker in the mass spectra of the at least one test ampholyte composition and the reference ampholyte composition; iii. normalizing the relative intensity of the base peak of the at least one marker to the maximum relative intensity of the base peak measured from the at least one test ampholyte composition or the reference ampholyte composition; iv. comparing the normalized relative intensities of the base peaks of the at least one marker in the at least one test ampholyte composition and the reference ampholyte composition; The method of claim 11 , comprising:

13. 13. The method of any one of claims 1 to 12, wherein the at least one marker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 markers with different m / z.

14. The method of any one of claims 1 to 12, wherein the at least one marker comprises 16 markers with different m / z.

15. 15. The method of claim 14, wherein the 16 markers include markers at m / z 280, m / z 319, m / z 329, m / z 347, m / z 373, m / z 375, m / z 376, m / z 431, m / z 504, m / z 506, m / z 508, m / z 520, m / z 534, m / z 562, m / z 906, and m / z 980.

16. The method of any one of claims 1 to 15, wherein the mass spectrometry comprises ion mobility quadrupole time-of-flight mass spectrometry (IMS-Q-ToF-MS).

17. The method of any one of claims 1 to 16, wherein the liquid chromatography comprises high performance liquid chromatography (HPLC).

18. 18. The method of claim 17, wherein the HPLC comprises a C4 silica column.

19. 19. The method of any one of claims 1 to 18, further comprising validating the at least one test ampholyte composition by generating imaged capillary isoelectric focusing (iCIEF) electropherograms of a reference protein using the reference ampholyte composition and the at least one test ampholyte composition, thereby generating at least one test electropherogram and one reference electropherogram.

20. 20. The method of claim 19, wherein the test ampholyte composition is verified if the at least one test electropherogram and the reference electropherogram are similar.

21. 21. The method of claim 20, wherein the similarity of the at least one test electropherogram and the reference electropherogram is determined by the number, size, or isoelectric point (pI) of peaks, or a combination thereof.

22. 22. The method of claim 20 or 21, wherein the similarity of the at least one test electropherogram and the reference electropherogram comprises having the same number of peaks.

23. 23. The method of any one of claims 20 to 22, wherein the similarity between the at least one test electropherogram and the reference electropherogram comprises a difference in the area under the peak of a region of the reference electropherogram and the at least one test electropherogram having a p-value of less than 0.

05.

24. 24. The method of claim 23, wherein the region comprises a major base peak, a region that is more acidic than the major base peak, or a region that is more basic than the major base peak.

25. The method of any one of claims 1 to 24, wherein the suitable activity comprises capillary isoelectric focusing (CIEF) or imaged capillary isoelectric focusing (iCIEF).

26. 26. The method of claim 25, wherein the iCIEF is used to characterize a protein drug product or drug substance.

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