EA047259B1 - LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LC-MS) METHODS FOR ANALYSIS OF AMPHOLYTE BATCH VARIATION - Google Patents

Description

Cross-reference to related applications

The invention claims the benefit of priority of 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.

Field of technology

This invention relates to methods for determining the charge characteristics of protein compositions and determining the characteristics of ampholytic compositions using liquid chromatography-mass spectrometry (LC-MS).

State of the art

Many proteins undergo secondary modifications that result in protein charge species such as deamidation, N-terminal pyroglutamate formation, aggregation, isomerization, sialylated glycans, fragmentation, and glycation of lysine residues. In some cases, these secondary modifications and the resulting charge species can impact protein binding, biological activity, patient safety, and shelf life. Tools such as isoelectric-focusing gel electrophoresis (IEF) and capillary equivalents such as capillary isoelectric focusing (CIEF) and imaged CIEF (iCIEF) can be used to analyze protein charge. These techniques are important for characterizing and monitoring the quality, purity, stability, and variability of therapeutic proteins such as protein-based drugs and drug substances. One such method, iCIEF, has significantly contributed to biopharmaceutical development due to its high resolution, minimal development time, reduced sample volume, and short turnaround times. These advantages ensure their application in all pharmaceutical processes, from cell culture development and optimization to commercial quality control (QC) and stability activities.

In CIEF and iCIEF, separation of proteins with different net charges is achieved by focusing the proteins in an ampholytic pH gradient using an applied electric field, separating the isoforms based on their intrinsic isoelectric charge. This separation method relies on the use of ampholytes to create a pH gradient when an electrical potential is applied. The protein particles then migrate to different regions of the capillary based on their isoelectric point, or pI value. However, lot-to-lot differences in the ampholyte compositions used to create the pH gradient in these methods can affect the assay results. Previous methods for characterizing ampholyte compositions for suitability for iCIEF and similar methods have relied on indirect methods, such as using ampholyte compositions to generate iCIEF electropherograms or similar readouts of a reference protein and comparing the resulting electropherograms obtained with different ampholyte compositions. However, these methods are expensive, time-consuming, and use large amounts of protein. There is thus a need in the art for improved methods for characterizing ampholyte compositions for their usefulness in methods such as iCIEF. The present invention satisfies this need by providing improved methods for the direct analysis of ampholyte compositions.

The essence of the invention

The present invention provides methods for identifying a test ampholyte composition with a suitable activity, comprising: (a) identifying at least one marker in at least one test ampholyte composition and a reference ampholyte composition using liquid chromatography-mass spectrometry (LC-MS); and (b) determining a degree of similarity or difference of at least one marker between the at least one test ampholyte composition and a reference ampholyte composition, wherein the at least one test ampholyte composition has a suitable 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 wherein the at least one test ampholyte composition has a suitable activity if the at least one marker has a high covariance and the level of the at least one marker is the same between the at least one test ampholyte composition and the reference ampholyte composition; thereby identifying a test ampholyte composition with a suitable activity.

In some embodiments of the methods of the present invention, step (a) comprises (i) determining an accurate mass/retention time (AMRT) measurement or collision cross section (CCS) for a plurality of components of at least one test ampholyte composition and a reference ampholyte composition; (ii) plotting the covariance of the AMRT or collision cross section measurements for the plurality of components of at least one test and reference ampholyte compositions using an S-plot; and (iii)

- 1 047259 selecting at least one component that differs between at least one test ampholyte composition and reference ampholyte compositions, wherein the difference comprises a covariance that is different from 0 on an S-plot; thereby identifying at least one marker for determining the suitability characteristics of the at least one test ampholyte composition. In some embodiments, the difference comprises a covariance that is less than 0 on the S-plot. In some embodiments, the at least one component comprises a covariance on the S-plot that is less than a 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 totality of components on the S-plot. In some embodiments, at least one test ampholyte composition has suitable activity if the level of at least one marker in the at least one test ampholyte composition and the reference ampholyte composition differs.

In some embodiments of the methods of the present invention, the difference in the level of at least one marker is 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 at least one marker.

In some embodiments of the methods of the present invention, step (a) comprises (i) determining an accurate mass/retention time (AMRT) measurement or a collision cross-section measurement for a plurality of components of at least one test ampholyte composition and a reference ampholyte composition; (ii) plotting the covariance of the AMRT or collision cross-section measurements for a plurality of components of at least one test and reference ampholyte compositions using an S-plot; and (iii) selecting at least one component that is similar between the at least one test ampholyte composition and the reference ampholyte compositions, wherein the similarity includes a covariance that is different from 0 on the S-plot; thereby identifying at least one marker for determining the suitability characteristics of the at least one test ampholyte composition. In some embodiments, the difference includes a covariance that is greater than 0 on the S-plot. In some embodiments, at least one component has a covariance on 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 totality of components on the S-plot. In some embodiments, at least one test ampholyte composition has suitable activity if the level of at least one marker in the at least one test ampholyte composition and the reference ampholyte composition is similar.

In some embodiments of the methods of the present invention, the similarity of the level of at least one marker comprises a level that is 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 to the normalized level of at least one marker.

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

In some embodiments of the methods of the present invention, step (b) comprises: (i) determining an LC-MS mass spectrum of at least one marker in at least one test ampholyte composition and a reference ampholyte composition, (ii) determining a relative intensity of a major peak of at least one marker in the mass spectra of at least one test ampholyte composition and the reference ampholyte composition, (iii) normalizing the relative intensity of the major peak of at least one marker to a maximum relative intensity of the major peak measured for at least one test ampholyte composition or the reference ampholyte composition, and (iv) comparing the normalized relative intensities of the major peak of at least one marker in at least one test and reference ampholyte compositions.

In some embodiments of the methods of the present invention, 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 with 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.

In some embodiments of the methods of the present invention, the mass spectrometry comprises ion mobility quadrupole time-of-flight mass spectrometry (IMS-Q-ToF-MS).

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In some embodiments of the methods of the present invention, the liquid chromatography comprises high performance liquid chromatography (HPLC). In some embodiments, the HPLC comprises a C4 silica gel column.

In some embodiments of the methods of the present invention, the methods further comprise validating at least one test ampholyte composition by generating a reference protein electropherogram using capillary isoelectric focusing imaging (iCIEF) using the reference ampholyte composition and at least one test ampholyte composition, thereby generating at least one test electropherogram and a reference electropherogram.

In some embodiments of the methods of the present invention, the tested ampholyte composition is considered validated if at least one test and reference electropherograms are similar. In some embodiments, the similarity of at least one test and reference electropherogram is determined by the number, size, or isoelectric point (pI) of peaks, or a combination thereof.

In some embodiments of the methods of the present invention, the suitable activity comprises capillary isoelectric focusing (CIEF) or capillary isoelectric focusing with imaging (iCIEF). In some embodiments, iCIEF is used to characterize a protein drug product or drug substance.

Brief description of graphic materials

Figures 1A, 1B show a pair of electropherograms analyzing a reference protein product using capillary isoelectric focusing with imaging (iCIEF) analysis. The x-axis shows time (in minutes) and the y-axis shows absorbance. Figure 1A uses ampholyte lot 1. Figure 1B shows a typical candidate ampholyte lot that produces a different electropherogram profile for the same reference protein than the ampholyte lot shown in Figure 1A. Differences in the electropherograms are circled.

Figure 2 is a graph showing the baseline interference based on the ampholyte gradient used in iCIEF. ICIEF was performed without a reference protein. Increasing the ampholyte concentration resulted in a decrease in the baseline drop. This change in peak profile was due to the ampholytes, not the proteins or the instrument.

Fig. 3 shows an electropherogram showing that the concentration of the pharmacolyte (ampholyte) leads to a change in the area of the region. A change in the concentration of the pharmacolyte by 2% caused a decrease in the area of region 1 by 1%.

Figures 4A, 4B show a series of electropherograms (Figure 4A) and a table (Figure 4B) showing the change in resolution in the ampholyte batches. Batches 7 and 2 are the most suitable candidates for statistical analysis of the batch most similar to batch 1.

Figures 5A-5C are a series of graphs showing the isoelectric point (PI or pI) marker testing of ampholyte lots. The peak resolution for each ampholyte lot was calculated to range from 7.0 to 7.05. Figure 5A shows a typical electropherogram with two peaks. Figure 5B shows how the resolution is calculated from the electropherogram. The resolution is calculated based on the migration (cycle) time (Rt) and the peak width (W). Figure 5C shows a typical electropherogram in which the peaks overlap by 0.15%, or a resolution of 1.5, which is the chromatographic minimum criterion.

Fig. 6 is a graph depicting the problem solved by the methods of the present invention.

Fig. 7 is a graph showing the workflow of LC-MS analysis of ampholyte.

Fig. 8A is a table showing the lots of GE Healthcare IEF carrier ampholytes tested for their molecular components.

Figure 8B is a Waters S-plot showing testing of ampholyte lots for changes in molecular composition rather than characteristics. Liquid chromatography mass spectrometry (LC-MS) was used to identify components of interest in ampholyte lots that had high lot-to-lot variation. The analysis was performed using UNIFI software with built-in data analysis and workflows. The S-plot shows the differences in the true mass/retention time (AMRT) relationship between ampholyte lots. AMRT pairs are plotted by covariance, with the magnitude of change shown on the x-axis and the correlation, i.e., the consistency of change, shown on the y-axis.

Figures 9A-9C show Waters S-plots comparing ampholyte lots characterized using Waters VION IMS QT ion mobility quadrupole time-of-flight mass spectrometry (LC-IMS-Q-TOF-MS). Figure 9A shows a comparison of lots 1 and 2. Figure 9B shows a comparison of lots 1 and 3. Figure 9C shows a comparison of lots 1 and 5. The Waters S-plot identifies similarities and differences between samples and provides a powerful visual filtering tool. Unique ampholyte components were selected for the library

- 3 047259 features, for example, were selected as the highlighted points on the S-graph of batch 1 compared to batch 5 (Fig. 9C).

Figure 10A is a table showing the library of ampholyte components characterized by m/z (mass to charge ratio) value and their normalized relative intensities in ampholyte lots 1 (the current lot used in the standard operating procedure, or SOP lot), 2, 3, 4, and 5. The relative intensities were normalized to the highest relative intensity for all ampholyte lots. Marker response values were evaluated and compared in Excel format.

Figure 10B shows iCIEF electropherograms analyzing the product of reference protein 1 using 5 different lots of ampholyte. From top to bottom: lot 1 (SOP), lot 3, lot 4, and lot 5. Differences in iCIEF profiles are circled.

Figure 10C shows iCIEF electropherograms analyzing the product of reference protein 1 using ampholyte lot 1 (top, SOP) and lot 2 (bottom). After analysis of the LC-MS results, lot 2 was analyzed and was predicted to be the lot most similar to lot 1 (see Figure 10A).

Fig. 11A is a graph showing the ion mobility filtered extracted ion chromatogram (XIC) of the m/z 906.261 marker for each of the indicated ampholyte lots. The XIC response is used for lot comparison.

Figure 11B is a trend plot showing markers in triplicates of a given ampholyte lot (x-axis indicates lot and replicate) 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 control.

Figure 12 shows the structures of three markers from the library used to characterize the ampholyte batches. The structures correspond to the markers with m/z values at the bottom. These are commercially available elucidation results.

Figure 13 is a graph comparing the current ampholyte testing process with the new ampholyte testing methods described herein. The current methods reduce analysis time, as it takes 4 hours to analyze a new lot of ampholyte by liquid chromatography-mass spectrometry (LC-MS). In addition, the new ampholyte testing methods provide for ampholyte characterization without consuming any protein product. Finally, the new ampholyte testing methods are applicable to any new ampholyte supplier. A new marker library can be created for each supplier using the ampholyte lot comparison methods described herein.

Detailed description of the invention

The present invention provides methods for identifying ampholyte compositions with suitable activity for desired downstream applications, comprising: (a) identifying at least one marker in at least one test ampholyte composition and a reference ampholyte composition using liquid chromatography-mass spectrometry (LC-MS); and (b) determining the degree of similarity or difference of at least one marker between the at least one test ampholyte composition and the reference ampholyte composition. In some embodiments, the markers are selected based on differences between the reference and test ampholyte compositions, and the at least one test ampholyte composition has suitable activity if the at least one marker differs between the at least one test ampholyte composition and the reference ampholyte composition. In some embodiments, the markers are selected based on the similarity between the reference and test ampholyte compositions, and at least one test ampholyte composition has a suitable activity if at least one marker is similar between the at least one test ampholyte composition and the reference ampholyte composition. The markers can be identified by a specific mass-to-charge ratio (m/z) measured by mass spectrometry, and the levels or markers can be determined by the relative intensity of the specified m/z peak, which can optionally be normalized. This normalization can be related to the maximum relative intensity measured for the specified m/z peak in the ampholyte compositions in the assay. The markers can be further characterized by retention time in a liquid chromatographic separation step.

Suitable steps for downstream applications include, but are not limited to, isoelectric focusing (IEF) gel electrophoresis and capillary equivalents such as capillary isoelectric focusing (CIEF) and imaging-assisted CIEF (iCIEF).

In some embodiments, the methods further comprise validating at least one test ampholyte composition by generating a reference protein capillary isoelectric focusing (iCIEF) imaging electropherogram using the reference ampholyte composition and at least one test ampholyte composition, thereby generating at least one test electropherogram and a reference electropherogram.

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The similarity of the test and reference electropherograms can be determined by comparing the number, size, area, resolution, and isoelectric point (pI) of peaks in the reference and test electropherograms, or a combination of all these characteristics.

Use of ampholytic compositions.

The description provides methods for determining the suitability of the ampholytic compositions for one or more subsequent uses.

Ampholyte compositions, sometimes referred to as pharmalytes (e.g., commercially available ampholytes from GE Healthcare), are used in a variety of protein separation methods based on isoelectric charge. Exemplary methods for which ampholyte compositions can be optimized using the methods described herein include, but are not limited to, isoelectric focusing (IEF) gel electrophoresis and capillary equivalents such as capillary isoelectric focusing (CIEF) and imaging CIEF (iCIEF).

As used herein, the term ampholytes refers to compounds containing both positive and negative charges that behave as zwitterions at and near their isoelectric point (pI) values. An ampholyte can act as an acid or a base, depending on the pH of the solution into which it is added. Ampholytes facilitate the formation of pH gradients, such as those seen in focusing in iCIEF and CIEF.

The isoelectric point (pI) is the pH at which a molecule carries no net electrical charge. For a protein, the isoelectric point is the pH at which the net charge of the protein is zero.

Isoelectric focusing (IEF) gel electrophoresis.

IEF is a charge-based protein separation method that involves adding an ampholyte solution to an immobilized pH gradient gel (IPG). IPGs contain an acrylamide gel matrix copolymerized with a pH gradient that can be produced by embedding carrier ampholytes into the acrylamide matrix. The resulting pH gradients are stable in an electric field except at highly alkaline (>12) pH values. The pH gradient for protein separation is fully established prior to addition of the protein analytes by initially exposing the solution to small molecules, such as polyampholytes, with different pI values by electrophoresis.

When an electric field is applied, a protein whose pH is below its isoelectric point (pI) will be positively charged and will therefore migrate toward the cathode (the negatively charged electrode). As it migrates through the gradient of increasing pH, the net charge of the protein will decrease until the protein reaches the pH region corresponding to its pI. At this point, it has no net charge and so migration ceases (since there is no electrical attraction to either electrode). As a result, the proteins are focused into sharp, stationary bands, with each protein located at a point in the pH gradient corresponding to its pI.

Capillary isoelectric focusing (CIEF) and visualized capillary isoelectric focusing (iCIEF).

Capillary isoelectric focusing (CIEF) and capillary imaging isoelectric focusing (iCIEF) are analytical techniques that utilize individual protein variants primarily based on the intrinsic net isoelectric (pI) charge of the protein. In CIEF and iCIEF, sample separation is accomplished by focusing protein samples in an ampholyte pH gradient using an applied electric field. Protein samples are pre-mixed with carrier ampholytes, additives, and pI markers. The protein samples are then separated in a capillary cartridge with electrolyte reservoirs at each end. One reservoir is filled with an acid (anolyte) and the other with an alkali (catholyte). The sample mixture is injected to fill the capillary column and a voltage is applied to the anolyte and catholyte reservoirs. This creates a pH gradient that separates and focuses the proteins (analytes) based on their pI along the capillary.

In CIEF, the focusing step is followed by a mobilization step in which the focused analytes are moved through the capillary to the capillary outlet and past a detector such as a UV or fluorescence detector. Mobilization methods include hydrodynamic or pressure mobilization, i.e., by introducing a gas into the capillary, and chemical mobilization, in which the anolyte or catholyte is replaced by another electrolyte solution with a high ionic strength or a different pH.

Unlike CIEF, which involves mobilizing protein samples through a detector, in iCIEF a full-column detector, such as a UV or fluorescence detector, monitors the entire process in the capillary in real time. This allows for close monitoring of the focusing step, followed by a true profile of the final charge variant. Thus, the advantages of iCIEF over other methods include high resolution, time-efficient

- 5 047259 execution time and low sample consumption, making it the preferred method for protein characterization in the biopharmaceutical industry.

Variability and ampholytic compositions.

The output of a CIEF or iCIEF protein assay may be an electropherogram. In an electropherogram, the x-axis contains the isoelectric point or mobilization time. The y-axis contains the detector reading, usually in absorbance units. Peaks in the electropherogram are sometimes called absorbance peaks. Variations in ampholyte compositions may result in variability in the output electropherograms.

Electropherograms of a reference protein generated using a test ampholyte composition may have one or more of the following changes compared to an electropherogram of the same reference protein generated with a reference ampholyte composition: (1) an enhancement of one or more peaks, (2) a decay of one or more peaks, (3) shifts in the pI or mobilization time of one or more peaks, (4) a change in the area under the peak of one or more peaks, and (4) changes in the resolution of one or more peaks.

Accordingly, the invention provides methods for determining whether a test ampholyte composition will produce an electropherogram of a reference protein similar to an electropherogram of the same reference protein produced by a reference ampholyte. In some embodiments, the methods comprise (a) identifying at least one marker in at least one test ampholyte composition and the reference ampholyte composition using liquid chromatography-mass spectrometry (LC-MS); and (b) determining a degree of similarity or difference of at least one marker between the at least one test ampholyte composition and a reference ampholyte composition, wherein the at least one test ampholyte composition has a suitable 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, and wherein the at least one test ampholyte composition has a suitable activity if the at least one marker has a high covariance and the level of the at least one marker is the same between the at least one test ampholyte composition and the reference ampholyte composition; thereby identifying a test ampholyte composition with a suitable activity.

The invention further provides methods for validating the activity of ampholyte compositions characterized using the methods described herein, comprising creating electropherograms using a reference ampholyte composition and at least one test composition and comparing the electropherograms to determine their similarity.

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

In some embodiments, similar electropherograms have the same or identical number of peaks (e.g., the number of peaks varies, being 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.

In some embodiments, the similarity between at least one test and reference electropherogram includes similar resolution.

As used herein, the term resolution or R is a measure of the quality of separation, such as the separation of a pair of absorption peaks in an electropherogram. Methods for calculating the resolution between two peaks are shown in Figs. 5A-5C. A resolution of 1.5 (0.15% mutual overlap) is considered the basic separation of two peaks and the criterion for the chromatographic minimum. The higher the resolution, the better the separation of the two peaks. Resolution is a combination of two factors: selectivity and separation efficiency. Selectivity takes into account the distance between the maxima of two peaks. Higher separation efficiency is achieved with narrower peaks. Therefore, narrow peaks and large distances between peaks give large R values. The difference in migration time or pI of two peaks (Rt1, _Rt2 ) and the width of the peak bases (W1, _W2 ) are used to calculate the resolution.

In some embodiments, the similarity of the test and reference electropherograms 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 of the test and reference electropherograms comprises a difference in resolution of less than 0.075. In some embodiments, the similarity of the test and reference electropherograms comprises a difference in resolution of less than 0.072.

In some embodiments, the similarity between the test and reference electropherograms includes one or more peaks in the region of the electropherogram with similar areas. In some embodiments,

- 6 047259 the difference in the area of one or more peaks in a particular region of the test and reference electropherograms has a probability value (p value) less than or equal to 0.05. Methods for calculating the area under the peak will be known to those skilled in the art or those of ordinary skill in the art and include, for example, fitting a curve equation to the peak and integrating to find the included area. In some embodiments, specific regions of the reference and test electropherograms whose peak areas are calculated may be defined by specific pI markers included in the analyte mixture. In some embodiments, specific regions of the reference and test electropherograms whose peak areas are calculated may be defined by the presence of a major peak common to all electropherograms. For example, an electropherogram may be divided into a major peak and regions that are acidic or basic relative to the major peak.

Significance can be determined by calculating a p-value, or probability value, which is the probability that the null model (e.g., that two peaks will have the same area by chance) will be correct. Methods for calculating p-values will be known to those skilled in the art and include Student's t-test, chi-square test, analysis of variance (ANOVA), Pearson correlation coefficient, and Bonferroni-Dunn. 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.

In some embodiments, the difference in the area of one or more peaks in a specific region of the test and reference electropherograms has a coefficient of variation (% RSD) less than or equal to 5%. Percent RSD is defined as the ratio of the standard deviation to the mean value and demonstrates the degree of variability with respect to the average value of the occupancy (e.g., the repeatability of the electropherogram). Methods for calculating RSD in percent are known to those skilled in the art. In some embodiments, a data set is reproducible if the % RSD is less than 5%. In some embodiments, a data set is reproducible if the % RSD is less than 4%, less than 3%, less than 2%, or less than 1%.

Characterization of ampholytic compositions using liquid chromatography-mass spectrometry (LC-MS).

Provided herein are methods for determining characteristics of ampholyte compositions using liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the methods include identifying at least one marker in at least one test ampholyte composition and a reference ampholyte composition using liquid chromatography-mass spectrometry (LC-MS); and determining a 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 may be identified by an MS mass-to-charge ratio (m/z), discussed below, and levels of the at least one marker determined by a relative intensity measured by the MS for each marker of a given m/z. In some embodiments, the methods include identifying a plurality of markers in at least one test ampholyte composition and a reference ampholyte composition using liquid chromatography-mass spectrometry (LC-MS); and determining the degree of similarity or difference of the plurality of markers between the at least one test ampholyte composition and the reference ampholyte composition.

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 with multiple components, while mass spectrometry provides the structural identity and levels of individual components with high molecular specificity and detection sensitivity.

Liquid chromatography and mass spectrometry are described in document EP 3143392, the contents of which are incorporated herein by reference.

As used herein, the term chromatography refers to a process in which a chemical mixture containing a liquid or gas is separated into its components by differential distribution of the chemical species as they flow around, over, and/or through a stationary liquid or solid phase. Liquid chromatography or LC refers to the process of selectively retardation of one or more components of a liquid solution as the liquid is uniformly permeated through a column of finely divided particles or through capillary channels. The retardation results from the partitioning of the components of the mixture between one or more stationary phases and a bulk fluid (i.e., the mobile phase) as the fluid moves relative to the stationary phase(s). Liquid chromatography includes, but is not limited to, reversed-phase liquid chromatography (RPLC), high-performance

- 7 047259 liquid chromatography (HPLC), high turbulence liquid chromatography (HTLC), and ultra-performance liquid chromatography. Retention time refers to the length of time that a particular analyte, such as a component of an ampholyte composition, is retained by a liquid chromatography substrate before elution.

As used herein, the term high performance liquid chromatography or HPLC refers to liquid chromatography in which the degree of separation is enhanced by passing a mobile phase under pressure through a stationary phase, usually through a tightly packed column.

As used herein, the term ultra-performance liquid chromatography or UPLC refers to liquid chromatography methods with increased speed, sensitivity, and resolution compared to HPLC. UPLC is generally applicable to particles with diameters less than 2 μm. Separation and quantification in UPLC are performed at extremely high pressures (up to 100 MPa).

Gas chromatography (GC) refers to a separation technique that uses a flow of gas through a column, such as a glass or metal column, to separate compounds based on volatility and interactions with a liquid stationary phase. The mobile phase, the carrier gas, is typically an inert gas such as helium or a non-reactive gas such as nitrogen.

Mass spectrometry is performed using a mass spectrometer that contains an ion source to ionize the sample and create charged molecules for further analysis. In various embodiments, the ampholytic compositions and their components can be ionized by any method known to a person skilled in the art. Ionization sources used in various MS techniques include, but are not limited to, electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, 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, thermal spray ionization/plasma spray ionization, surface enhanced laser desorption ionization (SELDI), inductively coupled plasma (ICP), particle beam ionization, and ion mobility separation (IMS). One skilled in the art will appreciate that the choice of ionization method may be determined based on the analyte being measured, the type of sample, the type of detector, the choice of positive mode versus negative mode, etc.

As used herein, the term mass spectrometry or MS refers to an analytical technique for identifying compounds based on their mass. MS refers to methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio (m/z). MS technology typically involves ionizing compounds to form charged particles (e.g., ions) and determining the precise mass of the ions divided by their charge, known as m/z. Compounds may be ionized and detected by any suitable means. A mass spectrometer typically includes an ionizer and an ion detector. Typically, one or more molecules of interest are ionized and the ions are then introduced into the mass spectrographic instrument, where a combination of magnetic and electric fields causes the ions to follow a path through space that depends on their mass (m) and charge (z). See, e.g., U.S. Patents 6,204,500; 6,107,623; 6,268,144; and 6,124,137.

MS can generate and detect both positive and negative ions. As used herein, the term ionization or ionizing refers to the process of generating an analyte ion having a net electrical charge of one or more electron units. Positive ions are ions that have a net positive charge of one or more electron units. Negative ions are ions that have a net negative charge of one or more electron units. In electron ionization or EI methods, analytes in the gas or vapor phase are interacted with a stream of electrons. The collision of electrons with the analyte results in the formation of analyte ions, 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 (e.g., ammonia) is interacted with electrons and analyte ions are formed from the interaction of the reagent gas ions and the analyte molecules. In fast atom bombardment or FAB, a beam of high-energy atoms (often Xe or Ar) strikes a non-volatile sample, desorbing and ionizing the molecules contained in the sample. The samples to be tested are dissolved in a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2-nitrophylyl octyl ether, sulfolane, diethanolamine, and triethanolamine. Selecting a suitable matrix for a compound or sample is an empirical process.

As used in this document, the term matrix-assisted laser desorption ionization or MALDI refers to methods in which a non-volatile sample is exposed to laser irradiation that desorbs and ionizes analytes in the sample in various ways.

- 8 047259 ionization, including photoionization, protonation, deprotonation, and cluster decay. In the case of MALDI, the sample is mixed with an energy-absorbing matrix, which facilitates the desorption of the 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 about 15,000 Daltons.

Surface-enhanced laser desorption ionization or SELDI refers to another method in which a non-volatile sample is exposed to laser radiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. In the case of SELDI, the sample is typically attached to a surface that preferentially retains one or more analytes of interest. As with MALDI, this process can also use an energy-absorbing material to facilitate ionization.

Electrospray ionization or ESI refers to a technique in which a solution is passed through a short length of capillary tube to the end of which a high positive or negative electrical potential is applied. The solution reaching the end of the tube evaporates (atomizes) into a jet or spray of very fine droplets of solution in a solvent vapor. This mist of droplets passes through an evaporation chamber that is gently heated to prevent condensation and evaporation of the solvent. As the droplets become smaller, the density of the electrical surface charge increases until the natural repulsion between like charges causes the release of ions as well as neutral molecules.

Atmospheric pressure chemical ionization, or APCI, is a mass spectroscopy technique similar to ESI; however, APCI produces ions through ion-molecule reactions that occur in a plasma at atmospheric pressure. The plasma is maintained by an electrical discharge between a nebulizer capillary and a counter electrode. The ions are then typically extracted into a mass analyzer using a series of differentially pumped skimmer stages. A counter-current of dry and preheated N2 gas can be used to improve solvent removal. Gas phase ionization in APCI can be more effective than ESI for analyzing less polar species.

As used herein, the term atmospheric pressure photoionization or APPI refers to a form of mass spectroscopy in which the mechanism for photoionization of a molecule M is the absorption of a photon and the emission of an electron to form molecular M+. Since the photon energy is typically slightly above the ionization potential, the molecular ion is less susceptible to dissociation.

As used herein, the term inductively coupled plasma or ICP refers to methods in which a sample is exposed to a partially ionized gas at a temperature high enough to atomize and ionize most elements.

As used herein, the term ion mobility spectrometry, sometimes also referred to as ion mobility separation or IMS, refers to an analytical chemistry technique that separates ions in the gas phase based on their interactions with the colliding gas and their masses. In a first step, ions are separated according to their mobility through a buffer gas on a millisecond scale using an ion mobility spectrometer. The separated ions are then introduced into a mass analyzer in a second step, where their mass-to-charge ratio can be determined on a microsecond scale.

As used herein, the term field desorption refers to methods in which a non-volatile test sample is placed on an ionization surface and an intense electric field is used to generate analyte ions.

As used herein, the term desorption refers to the removal of an analyte from a surface and/or the penetration of an analyte into the gas phase.

After ionization of the ampholyte composition or its components, the ions thus created can be analyzed to determine m/z. Suitable analyzers for m/z determination include quadrupole analyzers, ion trap analyzers, time-of-flight analyzers, Fourier transform ion cyclotron resonance (FTICR) analyzers, and Orbitrap spectrometers. The ions can be detected using one of several detection modes. For example, only selected ions can be detected using a selective ion monitoring (SIM) mode, or alternatively, multiple ions can be detected using a scanning mode such as multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).

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 such that only ions with a certain m/z value move along the length of the quadrupole, while all other ions are rejected. In this way, quadrupole instruments can act as both a mass filter and a mass detector for ions introduced into the instrument. In time-of-flight

- In the ToF method, ions are accelerated in a uniform electrostatic field by means of ground and repulsor electrodes. The kinetic energy is held constant and the ions move along a field-free ToF tube. Since the kinetic energy is constant (KE=1/2MV ^A 2), ions with a lower m/z will have a higher velocity compared to those with a higher m/z. Quadrupole time-of-flight or QTof mass spectrometry refers to a type of mass spectrometry using mass spectrometers that couple a quadrupole, which functions as a collision chamber, to a time-of-flight analyzer. This allows for simultaneous analysis of all ions with high resolution and high mass accuracy. In a typical QTof system, the sample is delivered by an in-line liquid chromatography system and ionized. The particle beam then passes through an ion guide and enters the quadrupole, which then enters the ToF analyzer. In some embodiments, the MS method may use tandem mass spectrometry, or MS/MS. In this method, a precursor ion (also called a parent ion) derived from a molecule of interest may be filtered in an MS instrument, and the precursor ion is subsequently fragmented to yield one or more fragment ions (also called daughter ions or product ions), which are then analyzed in a second MS procedure. By carefully selecting the precursor ions, only ions derived from specific analytes are passed to the fragmentation chamber, where they collide with inert gas atoms to form fragment ions. Because both precursor ions and fragment ions are formed reproducibly under a given set of ionization/fragmentation conditions, the MS/MS method can be an extremely powerful analytical tool. For example, a combination of filtration/fragmentation can be used to remove interfering substances and can be particularly useful in complex samples such as biological samples.

A mass spectrometer typically provides the user with an ion scan or mass spectrum; that is, the relative abundance of each ion with a given m/z in a given range (e.g., from 400 m/z to 1600 m/z). The mass spectrum can be related to the amount of an analyte, such as a component of an ampholyte composition, in a sample by numerous methods known in the art. For example, given that the sampling and analysis parameters are carefully controlled, the relative abundance, sometimes called the relative intensity, of a given ion can be compared to a table in which this relative abundance is converted to an absolute amount of the parent molecule. Alternatively, molecular standards can be analyzed with the samples and a standard curve can be constructed based on the ion signal generated by these standards. Methods for creating and using such standard curves are well known in the art, and one skilled in the art can select an appropriate internal standard. Numerous other methods for relating the amount of an ion to the amount of a parent molecule are well known to those skilled in the art.

When the ions hit the detector, they produce a pulse of electrons that is converted into a digital signal. The resulting data is transmitted to a computer, which displays the number of ions per unit time. The areas under the peaks corresponding to specific ions or the amplitude of such peaks are measured, and the area or amplitude is correlated with the amount of the component or marker of interest in the ampholyte composition. In some embodiments, the area under the curves or the amplitude of the peaks of the fragment ion(s) and/or precursor ions is measured to determine the amount of analytes with a given m/z value. As described above, the relative abundance, sometimes referred to as relative intensity, or response of a given ion can be converted to an absolute amount of the original analyte using calibration standard curves based on the peaks of one or more ions of an internal molecular standard. The absolute amount of the component of the ampholyte composition detected by LC-MS can then be converted to an absolute amount of the component that was present in the original sample.

The relative abundance or relative intensity can also be defined as a y-axis of the mass spectrum. In some embodiments, the amount of the marker can be quantified relative to the amount of the most abundant ion (main peak) of the mass spectrum. In some embodiments, the amount of the marker can be quantified using a response factor or a standard curve. In some embodiments, quantifying the amount of the marker comprises (1) including an internal standard in the sample, such as a reference ampholyte and/or a test ampholyte composition, and (2) normalizing the marker to the internal standard in the sample. In some embodiments, quantifying the amount of the marker further comprises (3) normalizing the marker to the maximum corrected amount of the marker measured for multiple ampholyte compositions. For example, the marker can be normalized to the maximum corrected response (i.e., relative intensity) of the marker measured for multiple lots of ampholyte from the same manufacturer. Internal standards include, but are not limited to,

- 10 047259 are limited to them, ¹³ C ₆ -carbamazepine, which elutes in the middle of the cycle and has a high ionization efficiency.

In some implementations, the internal standard response may be used to normalize the response values for each m/z to account for instrument drift.

In some embodiments, changing the retention time (RT), collision cross section (CCS), or both, and the m/z of the internal standard during the run can be used to set tolerances on the marker assignment parameters.

In some embodiments, the relative intensity of a marker or component of an ampholyte composition is normalized, for example, by the maximum relative intensity of that marker measured for several ampholyte compositions.

As used herein, the term accurate mass, sometimes referred to as measured accurate mass, is an experimentally determined mass using MS that allows determination of the elemental composition of an analyte, such as a component of an ampholyte composition. Accurate mass/retention time or AMRT refers to a combination of (1) the retention time of a component of an ampholyte composition during liquid chromatography and (2) the accurate mass of the component as measured by mass spectrometry.

As used herein, collision cross section or CCS data can be obtained using ion mobility experiments. It is the effective interaction area between an individual ion and a neutral gas molecule, which is related to ion characteristics such as chemical structure and size. It can be obtained using methods such as drift tube ion mobility measurements or traveling wave IMS. For example, multiple measurements are performed in different electric fields to calculate CCS values.

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

Suitable LC/MS instruments and systems include, but are not limited to, the BioAccord Biopharmaceutical LC/MS System (Waters), the Waters Acquity H-Class UPLC in combination with the Waters Vion IMS-Q-ToF-MS, the Agilent Ultivo LC/MS System, and the Orbitrap LC/MS System (ThermoFisher).

Determination of markers for ampholytic compositions.

The invention provides methods for identifying at least one marker in an ampholyte composition, comprising: (i) determining accurate mass/retention time (AMRT) measurements or collision cross-section measurements for a plurality of components of at least one test ampholyte composition and a reference ampholyte composition; (ii) plotting the covariance of the AMRT measurements for the plurality of components of the at least one test and reference ampholyte compositions using an S-plot; and (iii) selecting at least one component that is either different 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 determining suitability characteristics of the at least one test ampholyte composition.

Similarities and differences between the AMRT measurements of the components of the ampholyte composition 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. Covariance, as used herein, refers to the joint variability of two random variables. If large values of one variable generally correspond to large values of the other variable, and the same is true for small values (i.e., the variables tend to exhibit similar behavior), the covariance is positive. In the opposite case, when large values of one variable generally correspond to small values of the other (i.e., the variables tend to exhibit opposite behavior), the covariance is negative.

The covariance can be calculated using methods such as principal component analysis (PCA), orthogonal projection onto planetary structures - discriminative analysis (OPLS-DA), and Waters' S-plot, which will be familiar to those skilled in the art.

Principal component analysis, or PCA, is a statistical procedure that uses an orthogonal transformation to transform a set of likely correlated variables into a set of values of linearly uncorrelated variables, called principal components. This transformation is defined such that the first principal component has the largest possible variance (i.e., accounts for as much of the variability in the data as possible), and each subsequent component, in turn, has the largest possible variance, subject to a constraint that is orthogonal to the preceding components. Thus, PCA enables the reduction of large sets of multivariate data into uncorrelated

- 11 047259 variables known as principal components. PCA is limited in that it cannot provide information about individual groups of samples from the plot.

In some embodiments, the data may be further extracted using orthogonal projection on latent structures - discriminative analysis (OPLS-DA). This statistical analysis enables the identification of specific features (e.g., ampholyte species) that contribute to the overall differences observed between the two groups.

In some embodiments, the PCA and optionally OPLS-DA data can be viewed as an S-plot. An S-plot is a statistical way of displaying the differences between two groups of values. Thus, an S-plot compares two samples (e.g., with n=3 measurements, minimum). For example, pairs of AMRTs and/or CCS measurements of components from a reference and test ampholyte compositions can be plotted using S-plots. In an S-plot, the covariance, or amount of change, is plotted on the x-axis, and the correlation, i.e., the consistency of the change, is plotted on the y-axis. The farther a marker is from the origin of the x-axis, the greater its contribution to the variance between the groups, and markers located further along the y-axis represent higher confidence in the analytical result. A typical S-plot plots the covariance of pairs of AMRTs from at least one test ampholyte composition and a reference ampholyte composition. Each point on the S-plot represents a specific m/z with an associated RT and CCS value. Markers are plotted by consistency between replicates versus magnitude of change. The further from the origin of the x-axis, the greater the difference in intensity (or relative concentration) between samples. Differences between groups may be due to markers that are present in only one group or to markers that vary greatly in intensity between groups. The further from the origin of the y-axis, the more reliable the analytical results are when measured in triplicate. The S-plot can be used to identify which features vary most between batches, and these markers can be used to construct a screening library to characterize ampholyte batches.

In some embodiments, methods for identifying at least one marker for determining the suitability of an ampholyte composition include: (i) determining accurate mass/retention time (AMRT) measurements for a plurality of components of at least one test ampholyte composition and a reference ampholyte composition; (ii) plotting the covariance of the AMRT measurements for the plurality of components of the at least one test and reference ampholyte compositions using an S-plot; and (iii) selecting at least one component that has a difference between the at least one test ampholyte composition and the reference ampholyte compositions, wherein the difference is a covariance less than 0 on the S-plot; thereby identifying at least one marker for determining the suitability characteristics of the at least one test ampholyte composition. When the covariance is less than 0 on the S-plot, the paired AMRT values have a negative correlation between the reference and test ampholyte compositions. Accordingly, at least one marker is selected based on differences between the reference and test ampholyte compositions, and differences in the presence and/or level of at least one marker between the reference and test ampholyte compositions determine the suitability of the test ampholyte composition.

In some embodiments, such as those embodiments in which at least one marker is a marker of differences between a reference and a test ampholyte composition, wherein at least one component selected as the marker has a covariance on 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 totality of components on the S-plot.

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 a normalized level of at least one marker. For example, the reference ampholyte composition comprises a normalized level of at least one marker of 100%, and the test ampholyte composition comprises a normalized level of at least one marker of 30%. The markers can be characterized by mass to charge ratio (m/z), and the marker levels are determined by relative intensity normalized by maximum relative intensity, as described herein.

In some embodiments, methods for identifying at least one marker for determining the suitability of an ampholyte composition include (i) determining accurate mass/retention time (AMRT) measurements for a plurality of components of at least one test ampholyte composition and a reference ampholyte composition; (ii) plotting the covariance of the AMRT measurements for the plurality of components of the at least one test ampholyte composition and the reference ampholyte compositions using an S-plot; and (iii) selecting at least one component that is similar between the at least one test ampholyte composition and the reference ampholyte composition.

- 12 047259 ampholyte composition and reference ampholyte compositions, wherein the similarity comprises a covariance greater than 0 on an S-plot; thus identifying at least one marker for determining the suitability characteristics of at least one ampholyte composition being tested. When the covariance is greater than 0 on the S-plot, the paired AMRT values are positively correlated between the reference and test ampholyte compositions. Accordingly, at least one marker is selected based on the similarities between the reference and test ampholyte compositions, and the similarity in the presence and/or level of at least one marker between the reference and test ampholyte compositions determines the suitability of the test ampholyte composition.

In some embodiments, such as those embodiments in which at least one marker is a marker of similarity between a reference and a test ampholyte composition, at least one component selected as the marker has a covariance on 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 total number of components on the S-plot.

In some embodiments, the at least one test ampholyte composition has suitable activity if the level of at least one marker in the at least one test ampholyte composition and the reference ampholyte composition is similar. In some embodiments, the similarity of the level of at least one marker comprises a level that is 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 to the normalized level of at least one marker. For example, the reference ampholyte composition comprises a normalized level of at least one marker that is 100%, and the test ampholyte composition comprises a normalized level of at least one marker that is at least 90%. As a further example, the reference ampholyte composition has a normalized level of at least one marker that is 100%, and the test ampholyte composition has a normalized level of at least one marker that is at least 80%. The markers can be characterized by mass-to-charge ratio (m/z), and the marker levels are determined by relative intensity normalized by maximum relative intensity, as described herein.

The invention provides methods for determining a degree of similarity or difference of at least one marker between at least one test and reference ampholyte composition. In some embodiments, the methods comprise: (i) determining an LC-MS mass spectrum of at least one marker in at least one test ampholyte composition and a reference ampholyte composition, (ii) determining a relative intensity of a major peak for at least one marker in the mass spectra of at least one test ampholyte composition and a reference ampholyte composition, (iv) normalizing the relative intensity of the major peak of the at least one marker by the maximum relative intensity of the major peak measured based on 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 major peak of the at least one marker in the at least one test and reference ampholyte compositions.

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

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.

In some embodiments, the at least one marker comprises 16 markers with different m/z. In some embodiments, the 16 markers comprise markers with 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.

Suitable sets of markers with different m/z that can be used to characterize the ampholyte compositions being tested can be determined de novo relative to a reference ampholyte composition using the methods described herein. For example, by characterizing the reference and test ampholyte compositions using LC-MS, plotting AMRT pairs using an S-plot, and selecting at least one or a set of markers based on covariance.

Alternatively or in addition, the ampholyte compositions being tested may be characterized using the methods described herein by comparison with a predetermined library of markers that have previously been identified using the method described herein. For example, the relative intensities

- 13 047259 at least one or a set of markers of the ampholyte composition being tested can be determined using LC-MS and compared with a pre-determined reference or standard library of markers.

Numerous exemplary configurations, methods, parameters, etc. are described in this description. However, it should be understood that such description is not intended to be limiting with respect to the scope of the present invention, but is instead provided as a description of exemplary embodiments.

Examples

Example 1: Imaging Capillary Isoelectric Focusing (iCIEF) Protocol for Determining Protein Charge Variant Levels and/or Identity of Protein Products iCIEF is used for laboratory testing of drug substances, formulated drug substances, drug products, and materials in the manufacturing process for which written specifications have been established. When used for release testing according to specifications, iCIEF standard operating procedures (SOPs) must be followed and any deviations corrected. The following is a typical iCIEF SOP protocol, the result of which is affected by variation depending on the lot of ampholyte used in the SOP protocol.

Definitions.

iCIEF - visualized capillary isoelectric focusing.

pI is the isoelectric point, pH of the molecule, where the total charge is zero.

Region 1 (acidic) - a group of peaks that are relatively acidic compared to the largest peak of the iCIEF electropherogram.

Region 2 (neutral) - the main peak/peaks corresponding to the largest protein peak/peaks.

Region 3 (alkaline) is a group of peaks that are relatively basic compared to the largest peak of the iCIEF electropherogram.

Ampholytes are compounds that have both positive and negative charges and behave as zwitterions at and near their pI values. Ampholytes promote the formation of a pH gradient during focusing.

Transfer Time Measurement - An experiment performed to ensure proper sample introduction. The measurement is performed when starting the iCE3 instrument or installing a new cartridge.

Premix mode - protein samples are premixed with carrier pharmaceuticals, additives and pI markers.

Materials.

Safe-Lock Eppendorf tubing, 1.5 ml (VWR, Cat. No. 21008-959) or similar tubing made from the same materials of construction.

cIEF cartridge with FC coating (ProteinSimple, PN 101701).

Amber tubes, 10 ml, 22^75, with caps (thin partitions) (ProteinSimple, PN 045-139).

Polypropylene tube with molded insert 300 µl (ProteinSimple, PN 045-133).

iCE3 septum pack (300 µl caps) (ProteinSimple, PN 045-134).

Electrolytic pipette (ProteinSimple, PN 101788) or similar pipette made of the same construction materials.

Portable microinjector capillary, coated (ProteinSimple, PN 102694).

Micro tube, 5 ml, natural (Argos Technologies, cat. no. T2076) or similar tube made of the same construction materials.

Amicon Ultra-0.5 centrifugal filter with Ultracel-10 membrane (Millipore, cat. no. UFC501096).

Chemicals.

Water purified by Milli-Q system or commercially available, such as HPLC grade water (VWR, Cat# JT4218-3) or water for injection (WFI), (ApP Pharmaceuticals, Cat# 918510).

The corresponding reference standard of antibodies (current standard lot).

Urea (Sigma-Aldrich, BioXtra, cat. no. U0631).

3-10 pharmaceuticals (VWR Cat. No. 17-0456-01 or Sigma Cat. No. P1522-25ML), store at 2-8°C.

0.5% methylcellulose (MC) solution (ProteinSimple, PN 102505).

1% methylcellulose (MC) solution (ProteinSimple, PN 101876).

Electrolyte Kit (ProteinSimple, PN 102506):

100 ml 0.08 M H3PO4.

100 ml 0.1 M NaOH.

Marker pI pI 5.12 (ProteinSimple, PN 102224).

Marker pI pI 9.50 (ProteinSimple, PN 101996).

Equipment.

Microcentrifuge for test preparation.

- 14 047259

ProteinSimple iCE3 Charge Variant Analyzer.

VWR single channel pipettes (VWR #89130-554, 556, 560, 562, 566) or pipettes with similar ranges.

Sterile pipette tips (VWR) or tips appropriate for the pipettes used.

Positive displacement pipettes (Rainin No. MR-10, MR-100, MR-250, MR-1000).

Rainin positive displacement pipette tips (Rainin Nos. C-10, C-100, C-250, C-1000).

Eppendorf Repeater Plus Pipette (VWR, Cat. No. 21516-002).

Eppendorf Combitips pipette tips (VWR, cat. no. 21516-138).

Analytical balance.

Reagent solutions.

The volumes of reagent solutions can be adjusted as needed.

8M Urea Solution: To prepare 8M urea solution, weigh 2.4 g urea into a 5 ml conical tube. Add water slowly to approximately the 4 ml mark. Stir until the urea dissolves. Dilute with water to a final volume of 5 ml. Mix well. Prepare fresh samples daily.

Master Mix: Below is a single preparation that should be scaled up as needed. For the general iCIEF method, pI 5.12 and pI 9.50 are recommended as acid and base markers. The master mix should be mixed well and a fresh batch should be prepared daily.

Table 1

Basic mixture

Component Quantity 8M urea solution 50 µl 3-10 pharmaceuticals 8 µl 1% methylcellulose (MC) solution 70 µl Water 30 µl pI marker (acidic) 1 µl Marker pI (base) 1 µl

Procedure.

You can use the standard startup procedure to start iCE3.

Preparation of reference standard: Any current reference standard can be used to demonstrate the suitability of the system. The appropriate reference sample is diluted with water to a concentration of 2 mg/mL. The standards are mixed by gentle inversion and the tubes are spun for approximately 15 s in a microcentrifuge.

In a separate tube, mix 40 µl of the 2 mg/ml reference standard or product test preparation with 160 µl of the master mix. At least 2 tubes should be prepared for injections of the required system suitability. This preparation can be scaled up accordingly. Mix gently and swirl for approximately 15 s to allow the contents to settle to the bottom of the tube.

In-process sample preparation: Dilute the sample in-process to a concentration of 2 mg/mL with water. Mix by gentle inversion. Assemble the centrifugal filtration unit by placing the filter in a clean microcentrifuge tube. Pre-wet the filter membrane by pipetting 400 µL of water into the filter, ensuring the entire membrane is covered. Remove water from the filter by pipetting and discard the water in a waste container. Load 200 µL of the diluted 2 mg/mL sample into the pre-wetted filter. Close the centrifugal filtration unit by capping the microcentrifuge tube in the attached filter. Centrifuge the unit at 14,000 relative centrifugal force (rcf) for 15 min.

Remove the device from the centrifuge. Remove the filter from the device and invert it into a clean microcentrifuge tube. Remove the microcentrifuge tube containing the removed buffer. Once the filter is placed in a clean microcentrifuge tube, it will not be possible to cap it. Place the uncapped device with the inverted filter in the centrifuge and spin for 2 min at 1000 rpm. After centrifugation, the concentrated protein will be in the microcentrifuge tube. Remove the filter.

Reconstitute the concentrated protein in 180 µl of water. Mix gently and swirl for approximately 15 s to allow the contents to settle to the bottom of the tube.

Mix 40 µl of the filtered sample with 160 µl of the master mix. Mix gently and swirl for approximately 15 s to allow the contents to settle to the bottom of the tube.

Obtaining drug substance, formulated drug substance and/or drug product: If the sample concentration is less than 20 mg/mL, the procedure should be followed

- 15 047259 substitution of the buffer described above for the in-process sample in place of the protocol below for producing a drug substance, formulated drug substance, or drug product.

If the drug substance, formulated drug substance or drug product is an antibody, it must not be shaken at any stage of production.

Dilute the test article (drug substance, formulated drug substance, or drug product) with water to a concentration of 2 mg/mL. Mix by gentle inversion and spin the tubes for approximately 15 s in a microcentrifuge. In a separate tube, mix 40 µL of the 2 mg/mL test article formulation with 160 µL of the master mix. Mix gently and spin for approximately 15 s to allow the contents to settle to the bottom of the tube.

Preparation of the control: mix 40 µl of water with 160 µl of the master mixture. Mix gently and swirl for approximately 15 s to allow the contents to settle to the bottom of the tube.

Sample loading using the iCE3 instrument: Use the iCE3 premix mode. Transfer 150 µl of the sample supernatant (preparation of reference standard/standard solution, test sample and control) prepared as described above into a 300 µl polypropylene tube with a molded insert. Ensure that there are no air bubbles at the bottom of the tubes.

Place sample tubes in the Alcott 720 autosampler. Fill a 10 mL amber tube with approximately 8 mL of 0.5% methylcellulose (MC) and place in position D of the autosampler. Use fresh MC daily to prevent microbial growth. Insert the water tube into a fresh container of Milli-Q water and place over the instrument. Place the waste tube in the waste container.

Setting up and running a batch (iCE3): Open the CFR software. Click batch/data and select development. The Manage batch files window appears. Create a batch file using one of the following options:

(1) To create a new batch file, enter the batch file name and select the folder to save the batch. Select the number of input cycles and click new file.

(2) To start a new batch file using a previously saved batch file as a template, find the desired file in the left pane of the batch file management window. Click open file. Click save as, enter a new file name, and click OK.

In a batch, the initial injection should be a control. Test samples should be bracketed by at least five injections of the appropriate product reference standard. Three injections of the reference standard before the test sample and two after, for a total of five injections of the reference standard, are recommended.

Occasionally, missed injections or stray peaks may occur that interfere with the protein profile, resulting in atypical electropherograms (in terms of intensity, number and character of peaks). Repeat injections of control, reference standard and test article may be made within 24 h of the initial injection using at least five injections of reference standard as brackets, provided that the reference standard used is suitable for injection of the respective samples.

Set up the focusing period parameters for batch processing by entering a unique, descriptive file name for each injection. Enter the reference standard/standard solution designation, LIMS number, or other sample identifier for the sample ID. Number the tubes sequentially. Focusing period 1 (pre-focusing) uses the following settings: 1 min at 1500 V. Focusing period 2 (focusing) uses the following settings: 7 min at 3000 V. The wash duration is 90 seconds and the transfer delay time is 0.00 min.

Set the autosampler parameters:

Temperature control: Yes; set to 10°C.

Input buffer duration: default value.

Sample Input Duration: Default value.

Loading time: 6 sec.

Tube type: 2 ml with 300 µl insert.

Needle insertion depth: 48 mm.

The sample and buffer injection duration is determined during the transfer time measurement (system startup/cartridge installation check) and is based on the duration of time during which a plateau occurs. A duration of at least 10 s longer than the plateau duration is recommended.

Enter the following information in the sample conditions section:

Carrier ampholytes: 4% 3-10 pharmaceuticals.

Additives: 2 M urea, 0.35% MS.

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Low pl marker: pl value of the acid marker in the base mixture (5.12).

High pl marker: p! value of the alkaline marker in the base mixture (9.50).

Concentration [μg/μl]: 0.40.

Select the sample type (optional).

Please note that for concentration the control concentration is 0. The final sample concentrations in the process may vary depending on the dilution factor used.

Data transformation.

Data conversion can be performed using the CFR iCE software. The CFR software can be used to create electropherograms and set p! markers for the control, reference standard and test sample electropherograms. The CFR software can also be used to convert data to other formats such as Empower for additional analysis.

Filter suitability.

The electropherograms of the control, reference standard, and test sample injections should show both acidic (low) and alkaline (high) pl markers. The replicate injections of the reference standard should be similar in profile (i.e., intensity, number, and character of peaks). Each of region 1 and region 2 of at least five replicate injections of the reference standard should have an average % area result that meets the product specification of %RSD < 5%.

Verification of the reliability of the analysis.

The profile of the test article should be qualitatively similar to the typical electropherogram from the specific product's operating instructions for use in terms of intensity, number and character of peaks. There should be no peaks in the region of interest of the control input protein as specified in the relevant specific product's operating instructions.

Analysis of protein charge variants.

Results are reported to one decimal place. For the reference standard, the average % repeatability of areas 1, 2, and 3 for all injections is reported. The % RSD between all replicate injections of areas 1, 2, and 3 is also reported, and the average p! value of area 2 is reported for information only. For the test article (sometimes called the test sample), the data specified in the appropriate product specification or report are reported. If no report is available, the area % of areas 1, 2, and 3 is reported. The average p! value in area 2 is reported for information only.

Example 2: A change in the batch of ampholyte causes dispersion of the electropherogram.

Ampholytes are a critical reagent for iCIEF assays. However, different lots of ampholytes produce different electropherograms even when analyzing the same reference protein, leading to failure in quality control testing (cf. Fig. 1A and 1B).

Reference protein 1 was analyzed using iCIEF as described above for Example 1, using lot 1 of ampholyte (the current standard) and various lots of ampholytes tested. Table 1 below shows the GE ampholyte lots tested in this assay.

Table 1 3-10 Lots with pH of GE Healthcare Pharmalytes

GE Lots Analyzed (Current Standard)

iCIEF electropherograms of reference protein 1 generated under identical conditions but with different lots of ampholyte showed significant differences in profiles (e.g., cf. Fig. 1A with Fig. 1B, cf. electropherograms in Fig. 4A or 10B).

Changing the pharmacolyte concentration in the iCIEF assay showed that changing the ampholyte concentration by as little as 2% can cause changes in the electropherograms. For example, changing the ampholyte concentration from 4 to 6% resulted in a 1% decrease in the area of region 1 for reference protein 1 (Figure 3).

Differences in the ampholyte lot also affected the peak resolution in the iCIEF electropherograms. As shown in Fig. 5A-5C, the resolution of each ampholyte was calculated for pl ranging from 7.0 to 7.05.

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As shown in Fig. 4A-4B, different batches of ampholyte exhibit different peak resolutions in the range from 7.0 to 7.05.

Example 3: Characterization of an ampholyte batch using LC-MS and construction of a marker library.

Traditionally, ampholyte lots have been indirectly assessed by evaluating the performance of an iCIEF assay. The inventors have developed methods to directly characterize an ampholyte lot. These methods analyze the ampholytes themselves using LC-MS rather than analyzing the ability of the ampholyte lots to generate electropherograms of a known reference protein. This method characterizes the chemical composition of the ampholytes themselves using UPLC IMS-Q-ToF-MS and compares it to the performance of an iCIEF assay using a known reference protein.

GE Ampholyte Lot 1 was used to establish criteria in the standard operating procedures (SOP). However, a lot change was required. There were lot-to-lot differences in the different lots of GE Ampholytes, and these differences resulted in different electropherograms during repeated analyses of the same sample, leading to failed quality control tests. This can be seen in Example 2.

Thus, a method for ampholyte characterization using UPLC IMS-Q-ToF-MS for candidate lot evaluation was developed. The goal is to chemically characterize ampholyte lots and track components that differ between them. This provides a means for ampholyte characterization for future GE candidate lots and serves as a workflow that can be applied to other suppliers. Even without the ampholyte composition provided by the supplier, the method enables comparison of the candidate lot to the current lot using multivariate statistical analysis to assess differences.

Another advantage of using these methods is that the protein reference standard is not consumed, and the sample savings are 70:1 or the amount of sample consumed is reduced.

A schematic of the ampholyte testing methods is shown in Fig. 7. Briefly, ampholyte batches were analyzed in triplicate as is. Ampholyte batches were 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 full scan, which identifies components of the ampholyte batch with mass-to-charge ratios (m/z) in the range of 50 to 2000, although this range can vary. Components were identified within the IMS, m/z, and retention time (RT) tolerances. Differences in components identified by LC-MS were analyzed by multivariate analysis and plotted using Waters S-plots. Multivariate analysis identified differences between the ampholyte lots used to create a component library that can be used to assess differences between lots to predict which lots will behave similarly to a standard ampholyte lot in an iCIEF assay with a reference protein.

Fig. 8A shows batches of ampholytes that were analyzed by LC-MS to generate a component library.

Figure 8B shows a typical Waters S-plot for ampholyte LC-MS data. The Waters S-plot shows the differences between two ampholyte lots in the true mass/retention time (AMRT) relationship. AMRT pairs are plotted by covariance. The magnitude of the change is shown on the x-axis and the correlation or consistency of the change 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 concentration of that marker between lots. The further along the y-axis a marker component is located, the greater the consistency of the result between replicates. Differences between ampholytes may be due to components present or absent in one or more ampholyte lots and/or to changes in concentration of selected markers between lots.

UPLC IMS-Q-ToF-MS procedure.

Separations were performed on a Waters Acquity UPLC H-class system with a degasser, chiller, quaternary pump, and autosampler. A Waters BEHTM C4 2.1 x 100 mm, 1.7 μm particle size analytical column was operated at a flow rate of 0.2 mL/min. A 10 μL aliquot was 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 started with 95% A and 5% B held for 5 min, followed by a linear increase to 95% B after 35 min. This condition was maintained for 5 min before switching back to 95% A after 50 min. The column was kept under these conditions for an additional 10 min to re-equilibrate the column; The total cycle time was 60 min.

A Waters Vion IMS-Q-ToF-MS was used with positive electrospray ionization (+ESI) in the accurate mass screening mode in the MSe data mode. The mass spectrometer had the following parameters: capillary voltage (2.00 kV), source temperature (150°C), temperature

- 18 047259 desolvation gas (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.

Five lots of ampholytes (-1, -2, -3, -4 and -5) were evaluated and 500 µL of each lot were spiked with 25 µL of 1 µg mL ^-1 _13C6- carbamazepine, which was chosen as the internal standard due to its mid-run elution and high ionization efficiency. The response of the internal standard was used to normalize the response values for each m/z marker to account for instrument drift. In addition, the change in retention time (RT), collision cross section (CCS) and m/z of the internal standard during the run were used to set parameter-level tolerances for marker assignment. Each ampholyte sample was injected neat (n=3) from the same tube. Controls of the initial mobile phase compositions were also analyzed between each lot.

Data processing began with filtering and statistical analysis. Principal component analysis (PCA) reduces large multivariate data sets to uncorrelated variables known as principal components. PCA is limited in that it does not provide information about individual sample groups from the plot, but data can be further extracted using orthogonal projection onto latent structures of discrimination analysis (OPLS-DA). This statistical analysis identifies specific features (e.g. ampholyte species) that contribute to the overall differences observed between the two groups and can be thought of as an S-plot. An S-plot compares two samples (n=3 measurements, minimum); each point on the S-plot represents a specific m/z with an associated RT and CCS value. Markers are plotted by agreement between replicates versus magnitude of change. The further from the origin of the x-axis, the greater the difference in intensity (or relative concentration) between samples. Differences between groups may be due to markers that are present in only one group or to markers that vary greatly in intensity between groups. The further from the origin of the y-axis, the more reliable the assay results are when measured in triplicate. An S-plot was used to determine which characteristics differed most between batches and these markers were added to a screening library. The selected markers had to be present in all 3 dimensions of the ampholyte sample and should not be present in the controls. This library was then used to assess the presence or absence of specific markers for each batch, as well as presence at specific intensities. Markers that were suspected of having a greater impact on assay performance were further investigated by pattern elucidation and database searching for identification.

Results.

Figures 10B, 10C show the evolution of iCIEF profiles generated using Lots 1, 3, 4, and 5. iCIEF testing revealed that Lot 5 exhibited the most different results compared to Lot 1 (Figures 10B, 10C). In Figure 10B, the electropherograms become increasingly dissimilar to Lot 1 starting with Lot 1. The electropherogram generated using Lot 2 (Figure 10C) was most similar to that generated using Lot 1. Based on the marker library shown in Figure 10A, Lot 2 was predicted to have the greatest similarity in ampholyte components to Lot 1, the current standard ampholyte lot. As can be seen in Figure 10B, Lot 5 exhibited the most similar results compared to Lot 1. 10C, when Lot 2 was used to generate an iCIEF profile of Reference Protein 1, that profile was more similar to the profile of Reference Protein 1 of Lot 1 than any of the iCIEF profiles generated using Lots 3, 4, or 5 (Fig. 10B). Thus, the ampholyte component library can be used to directly identify ampholyte lots that generate iCIEF profiles similar to the standard lot, Lot 1.

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

The boxed points in Fig. 10C indicate the components that were selected for the component library used to characterize the ampholyte lots. Markers that were identified based on the S-plot were manually assessed to ensure that they were present in all three dimensions of the lot in which they were found and that they were unique to the ampholytes and not present in the control (Fig. 11B).

In addition, the extracted ion chromatogram (XIC) was monitored to ensure that a Gaussian peak was observed (Fig. 11A). All markers that met the criteria were added to the screening library. This library was then used to assess which markers were present, in which batches, and at what intensity. A heat map was generated representing

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Claims (20)

markers of interest, in which the normalized response of the internal standard in each batch was divided by the maximum normalized response across the 5 batches and multiplied by 100 to obtain the percentage (Fig. 10A). The trend observed in the heat map was confirmed by the iCIEF testing results. The greater the detected difference between the markers in Batch 1 compared to the other batches, the more the electropherogram deviated from the SOP. The markers that were suspected of having a greater impact on the assay performance were further investigated by structure elucidation and database searching for identification (Fig. 12) using the UNIFI scientific information system based on elemental composition calculation, structural database searching, and high collision energy data fragment matching (see Fig. 12 for structures, Fig. 11A for an example chromatogram used for identification). Conclusions. Characterization of lot-to-lot variation of ampholytes using UPLC IMSQ-ToF-MS enabled successful prediction of lot performance during iCIEF testing. This approach will improve the efficiency of selecting new lots of ampholytes, reduce the number of iCIEF tests, and conserve reference standard protein. The same workflow can be applied to many materials and suppliers if lot-to-lot variation is observed in the future. CLAUSE OF THE INVENTION 1. A method for identifying a test ampholyte composition with a suitable activity, wherein the suitable activity comprises obtaining an electropherogram of a reference protein similar to an electropherogram of the same reference protein obtained using a reference ampholyte composition, using capillary isoelectric focusing (CIEF) or capillary isoelectric focusing with imaging (iCIEF), wherein the method comprises: (a) identifying, by mass-to-charge ratio (m/z), at least one marker in at least one test ampholyte composition and in a reference ampholyte composition using liquid chromatography-mass spectrometry (LC-MS), wherein the at least one marker has a covariance other than 0, determined using an S-plot in an measured mass/retention time (AMRT) or collision cross-section for a plurality of components of the at least one test ampholyte composition and the reference ampholyte composition; and (b) determining a degree of similarity or difference of at least one marker between the at least one test ampholyte composition and a reference ampholyte composition, wherein the at least one test ampholyte composition has a suitable 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 wherein the at least one test ampholyte composition has a suitable 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; thus identifying the test ampholyte composition with suitable activity. 2. The method according to claim 1, characterized in that step (a) includes: i) determining the AMRT and/or impact cross-section for a set of components of at least one test ampholyte composition and a reference ampholyte composition; ii) plotting the covariance of the AMRT and/or collision cross-section measurements for a set of components from the at least one test and reference ampholyte compositions using an S-plot; 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 includes a covariance other than 0 on the S-plot; thus identifying at least one marker for determining the characteristics of a suitable activity of at least one ampholyte composition being tested. 3. The method according to claim 2, characterized in that at least one component contains a covariance on the S-graph 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 set of components on the S-graph. 4. The method according to claim 2 or 3, characterized in that at least one test ampholyte composition has suitable activity if the level of at least one marker in at least one test ampholyte composition and the reference ampholyte composition differs. 5. The method according to claim 4, characterized in that the difference in the level of at least one marker - 20 047259 includes 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 at least one marker. 6. The method according to claim 1, characterized in that step (a) includes: i) determining the AMRT and/or impact cross-section for a set of components of at least one test ampholyte composition and a reference ampholyte composition; ii) plotting the covariance of the AMRT measurements or collision cross-section for a set of components from the at least one test and reference ampholyte compositions using an S-plot; and 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 includes a covariance other than 0 on the S-plot; thus identifying at least one marker for determining the characteristics of a suitable activity of at least one ampholyte composition being tested. 7. The method according to claim 6, characterized in that at least one component includes a covariance on the S-graph that exceeds the covariance by 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 totality of components on the S-graph. 8. The method according to claim 6 or 7, characterized in that at least one test ampholyte composition has suitable activity if the level of at least one marker in at least one test ampholyte composition and the reference ampholyte composition is similar. 9. The method according to claim 8, characterized in that the similarity of the level of at least one marker includes a level that is 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 to the normalized level of at least one marker. 10. The method according to any one of claims 1 to 9, characterized in that the level of at least one marker is characterized by a relative intensity m/z in the mass spectrum. 11. The method according to claim 10, characterized in that step (b) includes: i) determining an LC-MS mass spectrum of at least one marker in at least one test ampholyte composition and a reference ampholyte composition, ii) determining a relative intensity of a major peak for at least one marker in the mass spectra of at least one test ampholyte composition and the reference ampholyte composition, iii) normalizing the relative intensity of the major peak of the at least one marker to the maximum relative intensity of the major peak measured based on the at least one test ampholyte composition or the reference ampholyte composition, and iv) comparing the normalized relative intensities of the major peak of the at least one marker in the at least one test and reference ampholyte compositions. 12. The method according to any one of claims 1 to 11, characterized in that at least one marker contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 markers with different m/z. 13. The method according to any one of claims 1-11, characterized in that at least one marker contains 16 markers with different m/z. 14. The method according to claim 13, characterized in that the 16 markers contain markers with 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. 15. The method according to any one of claims 1 to 14, characterized in that the mass spectrometry comprises ion mobility quadrupole time-of-flight mass spectrometry (IMS-Q-ToF-MS). 16. The method according to any one of claims 1 to 15, characterized in that the liquid chromatography comprises high performance liquid chromatography (HPLC). 17. The method according to claim 16, characterized in that the HPLC includes a column with silica gel C4. 18. The method of any one of claims 1 to 17, further comprising validating at least one test ampholyte composition by generating a reference protein capillary isoelectric focusing with imaging (iCIEF) electropherogram using the reference ampholyte composition and at least one test ampholyte composition, thereby generating at least one test electropherogram and a reference electropherogram. 19. The method according to claim 18, characterized in that the tested ampholytic composition is validated if at least one test and reference electropherogram are similar. 20. The method according to claim 19, characterized in that the similarity of at least one test and reference electropherogram is determined by the number, size or isoelectric point (pI) of peaks or their -