Imaging capillary isoelectric focusing to analyze protein variants in sample matrices

Abstract

The invention relates to imaging capillary isoelectric focusing for analyzing protein variants in a sample matrix. Embodiments of the present disclosure relate to methods, systems, devices, and kits corresponding to a method for analyzing a charge variant of a protein, such as vascular endothelial growth factor (VEGF-Trap).

Description

[0001] This application is a divisional application of an invention patent application with an application date of August 17, 2018, application number 201880053645.8, and invention name “Imaging capillary isoelectric focusing for analyzing protein variants in sample matrices”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application Serial No. 62 / 547,602, filed on August 18, 2017, which is incorporated herein by reference in its entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on August 15, 2018 is named REGE-005_001WO_ST25.txt and is 4,214 bytes in size. Technical Field

[0006] The field of the present disclosure relates to methods and systems for analyzing charge variants of proteins, such as VEGF Trap, in a sample matrix. Background Art

[0007] Analysis of charge variants is often required for various proteins used as biopharmaceuticals because such changes can affect drug activity, stability, and in some cases patient safety. Conventional methods used in the industry to identify and characterize charge variants include ion exchange chromatography, isoelectric focusing gel electrophoresis, and capillary isoelectric focusing. Imaging capillary isoelectric focusing has been found to be useful due to its high resolution, small sample volume, and fast run time. Therefore, methods and systems for determining charge variants of proteins such as VEGF Trap using imaging capillary isoelectric focusing would be beneficial. Summary of the invention

[0008] This paper describes methods and systems for charge variant analysis of various proteins. For example, the methods and systems can be used to analyze the charge variants of VEGF Trap. Rather than using the currently approved method for analyzing VEGF Trap, which groups bands 3-9 in isoelectric focusing gels to report charge heterogeneity distribution, the methods and systems described here generally use imaging capillary isoelectric focusing to report charge heterogeneity according to the percentage of charge variant isoforms, and group them into three different regions of the electropherogram. This reporting method can be more sensitive to changes that occur in the isoforms of VEGF Trap samples.

[0009] As mentioned above, embodiments of the present disclosure relate to methods, systems and apparatus for determining charge variants of proteins, and in particular, to an imaging capillary isoelectric focusing (iCIEF) assay for evaluating charge variants of proteins such as vascular endothelial growth factor (VEGF) blockers, hereinafter referred to as "VEGF-Trap." iCIEF is an alternative to the currently recognized isoelectric focusing (IEF) method for VEGF-Trap charge variant analysis.

[0010] The embodiments of iCIEF correspond to techniques for separating protein charge variants based on isoelectric point (pI). For example, in some embodiments, a protein sample is loaded onto a carrier ampholyte (e.g., Pharmalyte TM ), methylcellulose and a stabilizing additive (i.e., urea) on a separation capillary. A voltage is applied for a predetermined period of time, causing the carrier ampholyte to form a pH gradient within the capillary. In some embodiments, the voltage is applied for a longer second period of time corresponding to the "focusing" time. This causes the protein charge variants to migrate within the capillary until they reach a point where the overall charge of the variant is neutral (i.e., its pI).

[0011] In such embodiments, the capillary (which is coated with a fluorocarbon (FC)) is coupled to a digital (e.g., CCD) camera, thereby enabling direct detection and quantification of protein charge variants. Specifically, after a focus time, the CCD camera is configured to image the capillary (preferably in real time) to detect the protein within the capillary. In some embodiments, detection occurs at a wavelength of about 280 nm. Parameters in this technique include:

[0012] - Brand and concentration of ampholytes;

[0013] - the type and concentration of additives used; and

[0014] - Focusing time and sample concentration.

[0015] Aggregation and precipitation of proteins within the capillary are detrimental to the reproducibility of the electrophoretic pattern. For this reason, additives such as urea can be used to help stabilize and solubilize the proteins during focusing.

[0016] Therefore, in some embodiments, a method for analyzing charge variants of vascular endothelial growth factor VEGF-Trap is provided, and the method includes: loading a protein sample onto a separation capillary having a mixture of at least carrier ampholytes, methylcellulose, and a stabilizing additive; applying a first voltage for a first predetermined period of time so that the carrier ampholytes form a pH gradient within the capillary; applying a second voltage for a second predetermined period of time to focus the migration of the charge variants of the protein to their respective pIs; and detecting and quantifying the charge variants of the protein.

[0017] In such embodiments, detecting and quantifying charge variants comprises: measuring the absorbance of a plurality of charge variant isoforms; separating the plurality of charge variant isoforms into isolated regions comprising at least a first acid / acidic region (R1), a second neutral region (R2), and a third alkaline / basic region (R3); and determining the percentage of charge variant isoforms falling within each of regions R1, R2, and R3.

[0018] For such embodiments, image analysis for detection and quantification can be according to conventional methods and systems (eg, image analysis software).

[0019] In the embodiments summarized above, one and / or another of the following additional features / functionalities may be included (resulting in additional inventive embodiments), however, it should be noted that any one or more of these features may be different and still be within the scope of the invention, and the list provided below is only one embodiment:

[0020] - Detection and quantification of charge variants;

[0021] - Detection of charge variants occurs at a wavelength of approximately 280 nm;

[0022] - The protein concentration loaded onto the capillary was approximately 2.0 mg / ml;

[0023] - the stabilizing additive comprises urea;

[0024] - the amount of urea in the mixture comprises about 2M;

[0025] - the mixture includes about 0.35% methylcellulose;

[0026] - the first voltage comprises 1500V;

[0027] - the first predetermined time is about 1 minute;

[0028] - the second voltage comprises 3000V;

[0029] - the second predetermined time is about 7 minutes;

[0030] - the concentration of the protein loaded onto the capillary is between about 1.0 mg / ml and 8.0 mg / ml;

[0031] - the amount of urea in the mixture is greater than 0 M and less than about 8 M;

[0032] - the mixture includes between about 0.01% and about 0.35% methylcellulose, or any intermediate range;

[0033] - the first voltage is between about 1 V and about 3000 V or any intermediate range;

[0034] - the first predetermined time is between about 1 second and about 5 minutes or any intermediate range;

[0035] - the second voltage is between about 1 V and about 3000 V or any intermediate range; and / or

[0036] - The second predetermined time is between about 1 minute and about 14 minutes or any intermediate range.

[0037] In some embodiments, an iCIEF capillary configured for use in charge variant analysis of VEGF-Trap is provided, and the iCIEF capillary comprises a capillary configured to receive a protein and configured to have a mixture of carrier ampholytes, methylcellulose, and a stabilizing additive. The capillary may further comprise a fluorocarbon coating.

[0038] In some embodiments, an iCIEF kit configured for use in charge variant analysis of VEGF-Trap is provided, and the iCIEF kit comprises one or more capillaries configured to receive a protein and configured to have a mixture of carrier ampholytes, methylcellulose, and a stabilizing additive, wherein the capillaries comprise a fluorocarbon coating. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figures 1A to 1F The electropherograms of VEGF Trap using different ampholytes are shown. Figure 1A Amphoteric electrolytes are pharmalytes with a pI in the range of 3-10. TM ;exist Figure 1B Amphoteric electrolytes are pharmalytes with a pI in the range of 5-8. TM Pharmalyte with pI in the range of 8-10.5 TM combination of Figure 1C Amphoteric electrolytes are Servalytes with a pI in the range of 2-9. TM ;exist Figure 1D Amphoteric electrolytes are Servalytes with a pI in the range of 4-9.TM ;exist Figure 1E Amphoteric electrolytes are biolytes with a pI in the range of 3-10. TM and in Figure 1F Amphoteric electrolytes are pharmalytes with a pI in the range of 3-10. TM Pharmalyte with pI in the range of 6.7-7 TM combination.

[0040] FIG. 2A to FIG. 2E Comparison of electropherograms of VEGF Trap at different urea concentrations.

[0041] Figure 3 Comparison of electropherograms of VEGF Trap obtained using isoelectric focusing and imaging capillary isoelectric focusing methods.

[0042] FIG. 4A to FIG. 4G Shown are the analysis of VEGF Trap OFFGEL fractions (fractions 5-10) using isoelectric focusing and imaging capillary isoelectric focusing methods.

[0043] FIG. 5A to FIG. 5F Shown added FIG. 4A to FIG. 4G Electropherogram of VEGF Trap RS of VEGF Trap OFFGEL fractions (fractions 5 to 10) analyzed in .

[0044] Figure 6 Compare the electropherograms of blank spiked with independent markers and VEGF Trap.

[0045] Fig. 7A The imaged capillary isoelectric focusing electropherogram of the VEGF Trap RS sample assigned regions 1, 2 and 3 is shown.

[0046] Figure 7B The difference in electropherogram reporting between the isoelectric focusing method and the imaged capillary isoelectric focusing method is shown.

[0047] Figure 8 Data obtained using imaging capillary isoelectric focusing related to VEGF Trap stability are presented.

[0048] Fig. 9 Stability data obtained using imaging capillary isoelectric focusing of forced-degradation VEGF Trap samples are presented.

[0049] FIG. 10A to FIG. 10C Show Fig. 9 Statistical analysis of the imaging capillary isoelectric focusing data presented in .

[0050] Fig.11Data related to the linearity of the imaging capillary isoelectric focusing method are presented.

[0051] Fig.12 Comparison of the imaged capillary isoelectric electropherograms of three samples of ampholytes.

[0052] FIG. 13A to FIG. 13D Shown is a statistical analysis of imaged capillary isoelectric focusing data obtained from historical VEGF Trap samples. DETAILED DESCRIPTION

[0053] Described herein are methods and systems for charge variant analysis of various proteins such as VEGF Trap. VEGF Trap is a fusion protein comprising the sequences shown in Table 1. Rather than reporting the charge heterogeneity distribution by grouping bands 3-9 in an isoelectric focusing gel, as is currently recognized for analyzing VEGF Trap, the methods and systems described herein generally use imaging capillary isoelectric focusing to report charge heterogeneity based on the percentage of charge variant isoforms and group them into three distinct regions of the electropherogram. As previously described, this reporting method can be more sensitive to changes in isoforms of VEGF Trap samples.

[0054] Table 1 - VEGF Trap sequences

[0055]

[0056] The following abbreviations are used throughout this disclosure:

[0057] -iCIEF - Imaged Capillary Isoelectric Focusing

[0058] -IEF-Isoelectric Focusing

[0059] -pI-isoelectric point

[0060] -RS-Reference Standard

[0061] -DS-API

[0062] -DSI-API intermediates

[0063] -FDS-formulated API.

[0064] The method for analyzing charge variants of VEGF Trap generally includes: loading a protein sample onto a separation capillary containing a mixture of at least carrier ampholytes, methylcellulose, and a stabilizing additive; applying a first voltage for a first predetermined period of time so that the carrier ampholytes form a pH gradient within the capillary; applying a second voltage for a second predetermined period of time to focus the migration of the charge variants of the protein within the capillary so that the total load of the variants is neutral; and detecting and quantifying the charge variants of the protein.

[0065] The separation capillary can be loaded with VEGF Trap at a concentration ranging from about 0.5 mg / mL to about 2 mg / mL. For example, the separation capillary can be loaded with VEGF Trap at a concentration of about 0.5 mg / mL, about 1.0 mg / mL, about 1.5 mg / mL, or about 2 mg / mL. In some embodiments, the separation capillary is loaded with VEGF Trap at a concentration of about 1.0 mg / mL.

[0066] The amount of methylcellulose in the mixture can be in the range of about 0.01% to about 0.35%. For example, the amount of methylcellulose in the mixture can be about 0.01%, about 0.05%, about 0.10%, about 0.15%, about 0.20%, about 0.25%, about 0.30%, or about 0.35%. In some embodiments, the amount of methylcellulose in the mixture is about 0.35%.

[0067] As for the first voltage, it may be in the range of about 1 V to about 3000 V. For example, the first voltage may be about 1 V, about 100 V, about 500 V, about 1000 V, about 1500 V, about 2000 V, about 2500 V, or about 3000 V. In some embodiments, the first voltage is about 1500 V.

[0068] The second voltage may also be in the range of about 1 V to about 3000 V. For example, the second voltage may be about 1 V, about 100 V, about 500 V, about 1000 V, about 1500 V, about 2000 V, about 2500 V, or about 3000 V. In some embodiments, the second voltage is about 3000 V.

[0069] The first predetermined time may be in the range of about 1 second to about 5 minutes. For example, the first predetermined time may be about 1 second, about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 1 minute (60 seconds), about 1.5 minutes (90 seconds), about 2 minutes (120 seconds), about 2.5 minutes (150 seconds), about 3 minutes (180 seconds), about 3.5 minutes (210 seconds), about 4 minutes (240 seconds), about 4.5 minutes (270 seconds) or about 5 minutes (300 seconds). In some embodiments, the first predetermined time is about 1 minute (60 seconds).

[0070] The second predetermined time may be in the range of about 1 minute to about 14 minutes. For example, the second predetermined time may be about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, or about 14 minutes. In some embodiments, the second predetermined time is about 7 minutes.

[0071] Any suitable additive may be used in the mixture. In some embodiments, it may be beneficial to use urea as an additive. For example, it may be beneficial to include 2M urea in the mixture. Various reagents (ampholytes) may also be included in the mixture, as described below. In one embodiment, VEGF Trap is loaded into a capillary at a concentration of 1.0 mg / mL and analyzed using an imaging capillary isoelectric focusing method using a mixture of 0.35% methylcellulose, 2M urea, and 3% ampholytes with a pI of 3-10.

[0072] Reagents and Equipment. Table 2 below lists the reagents (ampholytes) and equipment used according to some embodiments of the present disclosure. Charge Variant Analyzer. Unless otherwise stated, VEGF-Trap Reference Standard (RSVITV-5) was used as the test article during method development and characterization.

[0073] Table 2 - Example reagents and other components used

[0074]

[0075] Examples of at least some embodiments:

[0076] Ampholyte screening was initially performed based on pI range and ampholyte source. Four ampholytes were obtained from three different sources, each covering a unique pI range. The following starting points were used to analyze the ampholytes:

[0077] -2.0 mg / mL protein concentrate;

[0078] -2M urea;

[0079] -0.35% methylcellulose;

[0080] - Prefocusing at 1500V for 1 minute; and

[0081] - Focus at 3000 V for 7 minutes.

[0082] Figures 1A to 1F Shown are electropherograms obtained using six ampholyte ranges with the iCE3 charge analyzer. The following ampholyte ranges were used:

[0083] Figure A-3-10Pharmalyte Figure B-5-8 / 8-10.5 Combination Pharmalyte

[0084] Figure C-2-9 Servalyt Figure D-4-9 Servalyt

[0085] Figure E - Bio-Lyte 3-10, 40% Figure F-3-10 Pharmalyte and 6.7-7.7 Pharmalyte 3-blend

[0086] Selected proteins. Amphoteric electrolytes with a pI range of 3-10 were selected as the overall profile of the iCIEF electropherogram because they are most similar to the electropherograms from the currently accepted VEGF-Trap charge variant analysis procedure (see e.g. Figure 3 IEF image shown).

[0087] Urea Optimization. According to some embodiments, process optimization further comprises varying the urea concentration (from no urea to 8M). FIG. 2A to FIG. 2E The effect of such varying urea concentrations on the electropherogram of VEGF-Trap RSVITV-5 samples is shown. Although a reduction in urea concentration generally improves resolution, it was found that increased urea (8M) resulted in reduced resolution. However, VEGF-Trap resolved under native conditions (without urea) has similar problems of reduced resolution and insufficient reproducibility. Therefore, when separated using 1M-3M urea concentrations, the overall peak shape and resolution of VEGF-Trap are comparable to each other.

[0088] Further experiments were performed using 2 M urea to optimize the concentration of ampholytes 3-10 and protein. Figure 3 The electropherogram obtained using 2M urea, 0.35% methylcellulose and 3% 3-10 ampholytes at 1.0 mg / mL protein concentration is shown. The iCIEF electropherogram was compared with another electropherogram and a tentative peak assignment was made ( Figure 3 ).

[0089] Method characterization. The total charge profile and peak shape obtained using the iCIEF assay are comparable to the IEF band profile (e.g. FIG. 2A to FIG. 2EAs shown). However, in order to further understand the IEF assay and to relate its banding pattern to the peak pattern obtained using iCIEF analysis, VEGF-Trap samples were subjected to OFFGEL classification. The individual charge variant fractions obtained from OFFGEL electrophoresis were analyzed using IEF and iCIEF assays. Agilent 3100OFFGEL classifier was used to classify VEGF-Trap reference standards according to their isoelectric points, and two methods, IEF and iCIEF, were used to analyze the isolated isotypes recovered as liquid fractions. VEGF-Trap RS was classified using an immobilized pH gradient strip (Immobilized pHGradient strip; IPG strip) with a pH of 6-9, covering the pI range of charge variants observed for VEGF-Trap. The detailed settings for the experiment and separation are as follows:

[0090] 1) Prepare a stock solution by combining 2.3 mL 50% glycerol, 230.4 μl IPG buffer (pH 6-11), and 16.64 ml water to give a total volume of 19.2 ml.

[0091] 2) Prepare VEGF Trap solution by adding 73 μg VEGF (4.2 mg) to 12 mL of stock solution and 3 mL of water.

[0092] 3) Prepare IPG strip rehydration solution in excess by combining 1.15 mL of water and 4.6 mL of stock solution.

[0093] 4) IPG gel strips (24 cm) with a pH in the range of 6-9 were arranged in every other lane of the two instrument trays, and 24-well frames were fixed in the appropriate positions above them. Standard OFFGEL kit protocol (see OFFGEL user manual: Agilent 3100 OFF GEL Fractionator Kit quick Start Guide, September 2010, 5th edition) was used for strip rehydration, antibody loading, and tray loading onto the instrument.

[0094] 5) Use a platform temperature of 20° C. A standard instrumental protein focusing method for a 24-well device was run using a constant current of 50 μA with a maximum voltage setting of 8000 V and a maximum power setting of 200 mW.

[0095] 6) After 34 h of fractionation, the run was stopped and similar wells from wells 3 to 12 in each lane were pooled and then exchanged into water and concentrated approximately 5-fold before analysis.

[0096] 7) The amount of antibody in each fraction was determined by measuring the absorbance at 280 nm (extinction coefficient of 1.15) on a Nanodrop (an instrument for determining fraction concentrations) and then multiplying the fraction volume by the concentration.

[0097] In short, 4.2mg VEGF-Trap RS was graded for 32 hours using 12 IPG strips; each fraction from each strip corresponding to the same pI range was collected and quantified after dialysis. After classification, a total of seven fractions (fraction 4-fraction 10) with sufficient recovery were analyzed using IEF and iCIEF. The fractions were analyzed in two ways: OFFGEL fractions (4-10) were analyzed separately using IEF and iCIEF assays (see Figure 4), and OFFGEL fractions (5-10) were added to VEGF-Trap RS, followed by IEF and iCIEF analysis of the added samples (see Figure 5). Table 3 lists the fractions recovered from the OFFGEL classification study and the corresponding amounts.

[0098] Table 3

[0099]

[0100] In addition to analyzing OFFGEL fractions independently using IEF and iCIEF, fractions with higher recovery (5-10) were spiked at a ratio of 1:0.1 (VEGF-Trap RS: fraction) and analyzed by two charge variant analysis methods. Figure 5 shows a set of electropherograms of VEGF-Trap RS spiked with VEGF-Trap OFFGEL fractions (5-10) analyzed using iCIEF (upper figure) and IEF (lower figure) assays. For each OFFGEL fraction spiked into RS, the corresponding control RS (unspiked) of the IEF and iCIEF assays was superimposed. The overlap of spiked and unspiked (RS+OFFGEL and RS) samples helps to visualize and understand the correlation of peak types between the IEF and iCIEF assays. In each figure, the peaks from the acidic fraction ( Figure 5A ) starts at the alkaline fraction ( Fig. 5F ) to emphasize the enhancement of the charged species corresponding to the spiked fraction.

[0101] From the analysis of the OFFGEL fractions, the correlation between the gel-based IEF banding patterns and the capillary-based iCIEF peak patterns is evident. From Figures 4 and 5 (together with the individual fraction analyses of the spiked fractions), it can be inferred that for VEGF-Trap, the charge isoform separation achieved by the currently accepted IEF gel method is comparable and similar to the peak patterns obtained using the capillary-based iCIEF method.

[0102] The charge variant distribution is reported using the iCIEF method. The currently recognized IEF method for VEGF-Trap charge variant distribution reports the charge variant percentage by grouping bands 3-9 of the IEF gel. Using, for example, myoglobin (an independent protein marker) as a guide to identify the number of bands based on the pI of myoglobin, the sum of the area percentages of bands 3-9 is calculated and reported. The current specification acceptance criterion (SPEC) for the IEF method is 82% (bands 3-9).

[0103] The new iCIEF assay used a similar approach, where an independent marker from ProteinSimple, pi 7.05 marker accession number 102226, was spiked into the iCIEF master mix (2 M urea, 0.35% methylcellulose, 3% 3-10 ampholytes). Figure 6 (Top) shows the iCIEF electropherogram of marker 7.05 spiked into the master mix, where bracketing pI 5.12 and pI 9.50 markers were used for calibration purposes. The bottom panel of Figure 5 shows the iCIEF electropherogram of VEGF-Trap RS, superimposed with a blank containing the spiked 7.05 marker, which will be used to identify peak 5 in the VEGF-Trap iCIEF sample profile. Marker peak 7.05 migrates at a pI between peaks 4 and 5, and it will be used to identify the main peak 6 in the cluster of main isoforms of VEGF-Trap (peaks 5, 6, and 7).

[0104] Instead of reporting peaks 3-9 as in the IEF method, the iCIEF method (according to some embodiments) reports the charge heterogeneity of VEGF-Trap samples according to the percentage of charge variant isoforms grouped into Region 1 (acidic), Region 2 (neutral), and Region 3 (basic). The cluster of three major peaks (peak numbers 5, 6, and 7) that migrate around the neutral pI range and are the most prominent isoforms in the VEGF-Trap iCIEF electropherogram are grouped into Region 2 (neutral). In the cluster of three peaks, a separate pI 7.05 marker spiked into a blank injection is used as shown in FIG7 to identify different isoforms corresponding to the major peak 5 that migrates to a specific pI. Region 1 (acidic) in the VEGF-Trap iCIEF sample is reported as a relatively acidic peak group compared to the cluster of three major peaks (peaks 5, 6, and 7) in the VEGF-Trap electropherogram. Region 3 (basic) in the VEGF-Trap iCIEF sample is reported as a relatively basic group of peaks compared to the cluster of three major peaks (peaks 5, 6, and 7) in the VEGF-Trap electropherogram.

[0105] The reporter approach using regions 1, 2, and 3 offers the advantage of allowing tighter control of charge variant isotypes by monitoring three regions (regions 1, 2, and 3) as opposed to the grouping of bands 3-9 of traditional IEF gel-based approaches. Figure 7B The difference between IEF report and the report according to the embodiment of iCIEF method disclosed herein is shown. In addition, the report according to region 1, 2 and 3 provides unique advantages for iCIEF assay method, i.e., compared with traditional methods, its sensitivity to charge variant isotype changes is much earlier. This makes the readout of iCIEF assay much more sensitive and better stability indication assay compared with previous IEF assay process. Table 4 below provides the example of the stability indication ability of the new grouping method adopted by iCIEF assay method in contrast with the traditional 3-9 report of IEF assay. By two kinds of reporting methods, i.e., regional grouping and peak 3-9 similar to IEF assay method, new iCIEF assay is used to analyze the VEGF-Trap stability sample accelerated and compare it with the historical results of IEF assay method.

[0106] As can be seen in Table 4 (below), the region 1, 2 and 3 grouping method is more sensitive and indicative of changes in the charge variant distribution of the VEGF-Trap samples. The IEF method showed a 2% decrease in the overall charge distribution of bands 3-9, and this change was comparable to the results of the iCIEF assay when the 3-9 peak method was used for grouping. However, it is obvious from Table 4 that for the VEGF-Trap samples subjected to accelerated stress at 25°C, an increase of 5% in region 1 (or acidic variants) and a concomitant decrease of about 5% in region 3 (basic variants) were observed using the iCIEF assay. This trend observed in the VEGF-Trap charge distribution in the iCIEF assay accurately reflects the nature of the changes that occur in the VEGF-Trap samples based on the complexity of their structure and charge pattern due to their different degrees of sialylation. The increase in acidic variants (region 1-highly sialic acid species) of the VEGF-Trap samples under accelerated thermal stress using the iCIEF assay reflects possible deamidation and aggregation. On the other hand, the use of traditional 3-9 band grouping by the IEF method masks subtle changes occurring in VEGF-Trap charge isoforms and fails to control for differently charged species, making it less sensitive than methods that detect subtle changes in charge heterogeneity of VEGF-Trap samples.

[0107] VEGF-Trap has 10 glycosylation sites. The polysaccharide chains connected to these sites are branched, and the end of each branch may or may not be negatively charged sugar monomer sialic acid. In the case of the presence of sialic acid groups at the ends of the polysaccharide chains, natural variation results in a collection of VEGF-Trap charge variants with a certain range of net charge. The ratio of these bands varies according to the abundance of the charged species present. Therefore, the new reporting method based on region 1 (severe sialylation), region 2 (moderate sialylation) and region 3 (minimum sialylation) to group various charged species makes iCIEF determination of the changes occurring in the sialic acid conversion (sialylform) of VEGF-Trap samples have a better response.

[0108] Table 4

[0109]

[0110] Stability indication capability of the iCIEF assay. Real-time stability samples of VEGF-Trap DP samples (kept at 2-8°C) were analyzed using 7 independent time points spanning a 24-month period; Table 5 (below) shows the data corresponding to this study. The historical IEF data for these VEGF-Trap samples are provided for reference and compared with the VEGF-Trap iCIEF data reported for regional groupings and 3-9 peaks. Under real-time storage conditions of 2-8°C, almost no significant changes in VEGF-Trap samples were observed based on historical IEF data, and similar trends were observed when reported using the iCIEF 3-9 peak method.

[0111] Table 5 - Real-time VEGF-Trap samples analyzed using iCIEF

[0112]

[0113]

[0114] Figure 8 A linear fit of the percentage of zones 1, 2, and 3 for VEGF-Trap over a 24 month period using iCIEF is shown. A small but steady increase in zone 1 and a decrease in zone 3 are evident from the graph.

[0115] Use new iCIEF assay method, use the VEGF-Trap DS sample of forced degradation to carry out additional analysis.For this research, use iCIEF method (region and 3-9) to analyze the thermal degradation VEGF DS sample that dilutes and applies stress in the time period of 15 days at 45 ℃ at the time point of 0,3,9 and 15 days.From Table 6 (below), it is obvious that when compared with the 3-9 peak report at an earlier time point of the forced degradation VEGF-Trap sample, when using the regional method grouping, the subtle increase of acidic charge variant (region 1) is observed for iCIEF assay method.Although 3-9% report shows 2% change of total charge distribution in 3-9 peak, under the same conditions, region 1 shows 7% increase, and region 3 shows 8% reduction with time change. Fig. 9 Shown is the electropherogram of a forced degradation VEGF-Trap sample using the iCIEF assay; the increase in acidic species is evident from the profile.

[0116] Table 6 - Percentage distribution of forced degraded VEGF-Trap samples using iCIEF (thermal stress) analysis

[0117]

[0118] The percentage distribution of the three regions of the VEGF-Trap stressed samples was statistically analyzed by comparing with the corresponding peak percentages of the VEGF non-stressed samples. FIG. 10A to FIG. 10C Statistical analysis of the heat stressed VEGF-Trap samples of iCIEF data is shown. Statistically significant changes were observed for regions 1 and 3, respectively.

[0119] Based on the characterization and optimization data of the iCIEF assay method, the assay parameters were derived and are presented in Table 7 (below). These method conditions were evaluated for linearity, accuracy, precision, and intermediate precision.

[0120] Table 7 - Key method parameters obtained from development and optimization experiments

[0121] Urea 2M Methylcellulose 0.35％ Amphoteric electrolyte 3-10 3％ VEGF-Trap 1.0mg / mL Pre-focus 1min at 1500V Focus 7min at 3000V

[0122] iCIEF assay quantification. Linearity of the iCIEF assay. Method linearity was assessed by a single analyst. Sample solutions were prepared at different protein concentrations of 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, and 2.0 mg / mL using the VEGF-Trap reference standard. In this experiment, the protein concentration in the sample matrix varied from 0.5 mg / mL to 2 mg / mL while keeping the other matrix components constant at 3% ampholytes 3-10 and 0.35% methylcellulose. The focusing time was also kept constant at 1+7 minutes. Table 8 summarizes the percentage distribution of regions 1, 2, and 3 for the VEGF Trap samples in the linear range of 0.5 mg / mL to 2.0 mg / mL. Fig.11 A linearity plot of the concentration of VEGF-Trap samples as a function of area counts is provided in FIG.

[0123] Table 8 - Linearity of iCIEF Assay Method

[0124]

[0125]

[0126] Based on regression analysis, the analysis showed acceptable linearity in the protein concentration range of 0.5 mg / mL to 2.0 mg / mL, with R2>0.99. In addition, the isoform distribution remained consistent in this same concentration range. This indicates that the analysis is able to provide consistent results for peak area and isoform distribution in the protein concentration range of 0.5 mg / mL to 2.0 mg / mL.

[0127] Accuracy of the iCIEF assay. The method accuracy was assessed based on the dilution ratio using linearity data compared to the nominal concentration of 1.0 mg / mL. The dilution recoveries based on the linearity data are shown in Table 9 below. The percent recovery was calculated using = (percentage of measured area / percentage of nominal area) × 100%.

[0128] Table 9 - Accuracy of VEGF-Trap iCIEF assay based on dilution ratio

[0129]

[0130] For VEGF-Trap samples, the recoveries based on the dilution ratios for the three regions were within 98%-101% in the protein concentration range of 0.5 mg / mL to 2.0 mg / mL.

[0131] Intermediate Precision Analysis of iCIEF Method Intermediate precision was evaluated on VEGF Trap RS samples by two separate analysts (A, B) using their respective reagent preparations and using two iCE3 Charge Variant Analyzer instruments over 4 days. The results of the analysis are listed in Table 10 below.

[0132] Table 10 - Results of intermediate precision analysis

[0133]

[0134]

[0135] The proposed VEGF-Trap iCIEF test method demonstrated acceptable precision when performed by different analysts using different reagent preparations. The overall RSD% for all three regions was calculated and was within 2% RSD.

[0136] Robustness of the iCIEF assay. Several elements of the robustness of the analytical method were evaluated by various experiments. These experiments are listed in Table 11.

[0137] Table 11 - Summary of method robustness experiments

[0138]

[0139] Solution stability prepared in the machine. Solution stability was assessed by preparing samples of reference standards in the sample matrix and analyzing the samples using iCIEF. After analysis at 10°C in a matrix consisting of 3% ampholytes 3-10, 0.35% methylcellulose and 2M urea, the samples were stored in the iCE3 instrument. The samples were analyzed again approximately 24 hours later. The isotype distribution for each analysis is presented in Table 12 below.

[0140] Table 12 - Results of stability of iCIEF samples prepared in the machine

[0141]

[0142]

[0143] Based on the range (maximum and minimum) observed at each time point, the absolute difference between the sample analyzed at T = 0 and the sample analyzed again at T = 24 hours was calculated. The absolute difference of the three regions was equal to or less than 0.5%. This indicates that the sample is stable in the matrix for up to 24 hours when stored at 10°C in the iCE3 charge variant analyzer.

[0144] Evaluation of ampholytes (3-10) samples. Several samples of 3-10 ampholytes from the same source were analyzed. The overall charge variant profiles of VEGF-Trap samples analyzed using different samples were comparable. For some ampholyte samples, slight differences in the peak shape of the electropherogram profile were observed in region 1, however, the percentage distributions were similar and within the assay fluctuation range. Fig.12 Representative electropherograms of three ampholyte samples are shown.

[0145] Example - Analysis of Historical VEGF-Trap Release Samples (DS, FDS and DSI) Using the New iCIEF Assay

[0146] To further establish the robustness of the iCIEF assay, iCIEF using two ampholyte samples was used to analyze a total of 37 unique historical VEGF-Trap samples that were not related to lineage. The analysis included 15 VEGF-TrapDSI (drug intermediate, aqueous buffer solution, pH 6.2, containing 5mM sodium phosphate, 5mM sodium citrate and 100mM sodium chloride), 10 VEGF-Trap DS samples (drug SPEC C701, aqueous buffer solution, pH 6.2, containing 10mM sodium phosphate) and 12 VEGF-Trap FDS samples (prepared drug, SPEC C713 aqueous buffer solution, pH 6.2, containing 10mM sodium phosphate, 40mM sodium chloride, 0.03% (w / v) polysorbate 20 and 5% (w / v) sucrose). These VEGF-Trap samples were analyzed using the iCIEF assay.

[0147] Ampholytes are mixtures of different homologues of amphiphilic compounds between 3 and 10 in the scope of isoelectric points, which contribute to the pH gradient established under the influence of the electric field. The ampholytes 3-10 used in the iCIEF assay method are purchased from a source, and they are usually produced in batches. Based on the advice of suppliers and our working knowledge of the iCIEF assay of other proteins, it is observed that the slight difference between different samples and this slight difference is inevitable. Therefore, in order to establish the robustness of new iCIEF assay in different ampholyte samples, two ampholyte samples were analyzed. The regional grouping method (R1, R2 and R3) proposed is used and the VEGF samples of DS, DSI and FDS product stages are also analyzed based on the 3-9 peak grouping similar to the IEF assay method. FIG. 13A to FIG. 13C Statistical analysis of the ampholyte samples for VEGF-Trap DS, DSI and FDS samples is shown as regions 1, 2 and 3 and peaks 3-9. Table 13 provides data for one ampholyte sample corresponding to the 37 VEGF-Trap samples. Tables 14 to 16 provide the complete data set for the 37 samples.

[0148] Table 13 - Historical VEGF-Trap DSI, DS, and FDS samples analyzed using the iCIEF assay process

[0149]

[0150]

[0151] Table 14 - DSI batches analyzed by Pharmalyte samples

[0152] VEGF Trap DSI Sample R1％ R2％ R3％ 3 to 9 peak% 1 30.2 43.1 26.7 87.9 2 27.8 44.4 27.8 88.8 3 28.0 44.5 27.6 88.9 4 27.9 44.4 27.8 88.7 5 28.8 44.8 26.4 85.3 6 27.5 44.3 28.2 87.1 7 23.7 41.4 34.9 89.7 8 26.9 42.9 30.2 88.4 9 28.5 45.1 26.4 89.3 10 27.6 43.9 28.4 88.7 11 28.3 44.9 26.8 88.3 12 28.1 44.6 27.3 87.8 13 28.2 43.7 28.1 87.8 14 27.6 43.5 28.9 87.9 15 29.3 44.7 26.0 89.2

[0153] Table 15 - DS batches analyzed by Pharmalyte samples

[0154]

[0155]

[0156] Table 16 - FDS batches analyzed by Pharmalyte samples

[0157]

[0158] Paired analysis was performed based on data from ampholyte samples collected from regions 1, 2, and 3 and 3-9 grouping methods ( Fig.13D ). The data from the paired analysis were used to compare the mean values ​​between the two ampholytes and to assess any differences in the assay reports that may be observed due to inherent differences in the ampholyte samples. Based on the data, it can be inferred that the maximum observed mean difference between samples of the three regions R1, R2, and R3 is less than 0.1% when reported by region. In addition, based on the p-values, it can be concluded that the percentage distribution between the two ampholyte samples in regions 1, 2, and 3 is not statistically significant using the regional method.

[0159] However, when the VEGF-Trap datasets for DS, DSI, and FDS samples were analyzed using a 3-9 peak grouping approach similar to the IEF assay, statistically significant differences were noted between some ampholyte samples. For example, between some samples, the average difference was as high as 4.8% when reported according to peaks 3-9 of the iCIEF assay. FIG. 13A to FIG. 13D An iCIEF profile obtained using different ampholyte samples is shown, and it is evident from the images that the fluctuations observed between ampholyte samples are confined to the acidic region and the fluctuations are masked by grouping regions 1, 2, and 3. This makes the region reporting method of iCIEF determination a robust and reproducible method.

[0160] The data from DSI, DS, and FDS sample analysis using ampholyte samples based on percentage region 1, 2, and 3 groupings make the iCIEF assay a more robust assay method.

[0161] Therefore, the iCIEF systems, methods and devices presented in the present disclosure quantify the charge variant profiles of VEGF-Trap APIs, API intermediates, formulated APIs and drug products. Such embodiments can be used to replace the currently approved gel-based IEF method for charge heterogeneity analysis of VEGF-Trap.

[0162] VEGF-Trap charge variants fractionated using the OFFGEL 3100 fractionator demonstrated correlation between peaks obtained with the capillary-based iCIEF assay method and bands obtained with the gel-based IEF assay process. Direct comparison of individual iCIEF peaks with IEF bands for VEGF-Trap charge variants was achieved by separate analysis of OFFEGEL electrophoresed VEGF-Trap fractions and by the spike waveforms in the study. The study confirmed that the new iCIEF assay process was able to resolve all charge variant isoforms previously resolved using the gel-based IEF method in an equivalent and more precise manner.

[0163] Therefore, some embodiments of this reporter method based on the percentage distribution of regions 1, 2, and 3 disclosed herein allow for control of all VEGF-Trap isoforms and make the assay more sensitive, making it a robust stability-indicating assay.

[0164] The present invention also describes a capillary used with the iCEF method. Generally speaking, the iCIEF capillary is configured for use in the charge variant analysis of VEGF-Trap and includes a capillary configured to receive a protein and configured to have a mixture of carrier ampholytes, methylcellulose, and a stabilizing additive. The capillary may also include a fluorocarbon coating.

[0165] In some embodiments, an iCIEF kit configured for use in charge variant analysis of VEGF-Trap is provided, and the iCIEF kit comprises one or more capillaries configured to receive a protein and configured to have a mixture of carrier ampholytes, methylcellulose, and a stabilizing additive, wherein the capillaries comprise a fluorocarbon coating.

[0166] Although various embodiments of the present invention have been described and illustrated herein, it is easy for a person of ordinary skill in the art to envision various other means and / or structures for performing functions and / or obtaining one or more of the results and / or advantages described herein, and such changes and / or modifications are each considered to be within the scope of the embodiments of the present invention described herein. More generally, it will be readily understood by those skilled in the art that all parameters, amounts, percentages, concentrations, sizes, materials, and configurations described herein are examples, and actual parameters, amounts, percentages, concentrations, sizes, materials, and / or configurations will depend on the specific application using the teachings of the present invention. It will be recognized by those skilled in the art that many equivalents of the specific embodiments of the present invention described herein can be determined using routine experiments alone. Therefore, it should be understood that the aforementioned embodiments are presented only by way of example. The embodiments of the present invention disclosed herein can be implemented in other ways different from the specific description and claim protection. The embodiments of the present invention disclosed herein also include separate features, systems, products, materials, kits, and methods described herein. In addition, any combination of two or more such features, systems, products, materials, kits, and methods is also inventive (if they do not contradict each other). Some embodiments may be distinguished from the prior art by the specific absence of one or more features / elements / functions (ie, claims directed to such embodiments may include a negative limitation).

[0167] In addition, as mentioned, various inventive concepts can be implemented as one or more methods. The actions performed as part of the method can be ordered in any suitable manner. Therefore, embodiments can be constructed in which the actions are performed in a different order than shown, which can include performing some actions simultaneously, even if these actions are shown as sequential actions in the illustrative embodiments.

[0168] Any and all references to publications or other documents presented anywhere in this application are incorporated herein by reference in their entirety. In addition, all definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

Claims

1. An imaging capillary isoelectric focusing (iCIEF) method for analyzing charge variants of vascular endothelial growth factor trap (VEGF-Trap) protein, the method comprising: loading a VEGF-Trap protein sample onto a separation capillary comprising a mixture of at least carrier ampholytes, methylcellulose, and urea; applying a first voltage for a first predetermined period of time such that the carrier ampholyte forms a pH gradient within the capillary; applying a second voltage for a second predetermined period of time to focus the migration of the charge variant of the VEGF-Trap protein within the capillary so that the overall charge of the charge variant is neutral; as well as Detecting and quantifying the charge variants of the VEGF-Trap protein, comprising: (i) measuring the absorbance of multiple charge variant isoforms; (ii) separating the plurality of charge variant isoforms into isolated regions comprising at least a first acid / acidic region (R1), a second neutral region (R2), and a third basic / alkaline region (R3), wherein the second neutral region (R2) comprises three major peaks identified relative to the independent marker peaks; and (iii) determining the percentage of charge variant isoforms falling within each of regions R1, R2, and R3.

2. The method of claim 1, wherein detection of charge variants occurs at a wavelength of 280 nm.

3. The method of claim 1, wherein the concentration of the protein loaded onto the separation capillary is 2.0 mg / ml.

4. The method of claim 1, wherein the amount of urea in the mixture comprises 1M to 3M.

5. The method of claim 1, wherein the mixture comprises 0.35% methylcellulose. The method of claim 1 , wherein the first voltage comprises 1500V. The method of claim 1 , wherein the first predetermined time is 1 minute. The method of claim 1 , wherein the second voltage comprises 3000V.

9. The method of claim 1, wherein the second predetermined time is 7 minutes.

10. The method of claim 1, wherein the concentration of the protein loaded onto the separation capillary is between 1.0 mg / ml and 8.0 mg / ml or any intermediate range.

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