EA041923B1 - CAPILLARY ISOELECTRIC FOCUSING WITH DETECTING DIRECTLY IN THE CAPILLARY FOR THE ANALYSIS OF PROTEIN VARIANTS IN THE SAMPLE MATRIX - Google Patents

Description

Cross-reference to related applications

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

Sequence listing

The currently pending application contains a sequence listing that was submitted in ASCII format via EFS-Web and incorporated herein in its entirety by reference. The specified ASCII copy, created on August 15, 2018, is named REGE005_001WO_ST25.txt and is 4214 bytes in size.

Technical field

The scope of the present disclosure encompasses methods and systems for analyzing charge variants of proteins, such as VEGF Trap, in a sample matrix.

State of the art

The analysis of different charge variants is often desirable for various proteins used as biopharmaceuticals, since such changes can affect the activity, stability and in some cases the safety of the patient. Common methods used in the industry to identify and characterize charge-different variants include ion exchange chromatography, isoelectric focusing gel electrophoresis, and capillary isoelectric focusing. Capillary isoelectric focusing with detection directly in the capillary has been found useful due to its high resolution, reduced sample size, and fast turnaround time. Accordingly, methods and systems using capillary isoelectric focusing with detection directly in the capillary to detect different charge variants for proteins such as VEGF Trap would be useful.

The essence of the invention

This document describes methods and systems for the analysis of different charge variants of different proteins. For example, the methods and systems may be applicable to the analysis of different charge variants of VEGF Trap. Instead of providing results on the distribution of different charge variants by grouping bands 3-9 in an isoelectric focusing gel, which is the currently approved method for assaying VEGF Trap, the methods and systems described herein typically use capillary isoelectric focusing with direct detection. in the capillary to give a conclusion about the charge heterogeneity in percentage of isoforms of different charge variants, and group them into three different regions of the electrophoregram. This reporting approach may be more sensitive to changes that are observed in isoforms of VEGF Trap samples.

As indicated above, embodiments of the present disclosure are directed to methods, systems, and devices for detecting charge-different protein variants, and in particular, capillary isoelectric focusing with direct-in-capillary detection (iCIEF) analysis to assess the charge dispersion of proteins such as an inhibitor vascular endothelial growth factor (VEGF), hereinafter referred to as VEGF-Trap. iCIEF is an alternative to the currently approved isoelectric focusing (IEF) based method for the analysis of charge differing VEGF-Trap variants.

Embodiments of iCIEF correspond to techniques that separate charge-different protein variants based on their isoelectric point (pI). For example, in some embodiments, a protein sample is loaded into a separation capillary containing a mixture of carrier ampholyte (eg, Pharmalyte™), methylcellulose, and a stabilizing agent (ie, urea). The voltage is applied for a predetermined period of time, causing the carrier ampholyte to form a pH gradient within the capillary. In some embodiments, voltage is applied for a second, longer period of time corresponding to focus time. This causes the different charge protein variants to migrate within the capillary until reaching a point where the overall charge of the variants is neutral (i.e., their pI).

In such embodiments, the capillary (which is coated with fluorocarbon (FC)) is connected to a digital (eg, CCD) camera that allows direct detection and quantification of charge-different protein variants. Specifically, after the CCD focusing time, the camera is set to image the capillary (preferably in real time) to detect the protein within the capillary. Detection, in some embodiments, occurs at a wavelength of approximately 280 nm.

The parameters of this technique include the brand and concentration of ampholyte, the type and concentration of additives used, and the focusing time and concentration of the sample.

Protein aggregation and precipitation within the capillary damages the reproducibility of the electrophoregram. For this purpose, additives such as urea can be used to help stabilize and solubilize the protein as it is in focus.

Accordingly, in some embodiments, a method is provided for assaying charge-different variants of vascular endothelial growth factor VEGF-Trap, and the method comprises loading a protein sample into a separating capillary that has a mixture of at least a carrier ampholyte, methylcellulose, and a stabilizing additive, 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 differing protein variants to their respective pI and detecting and quantifying the charge differing protein options.

In such embodiments, detection and quantification of charge differing variants includes measuring absorbance of multiple isoforms of charge differing variants, separating the plurality of isoforms of charge differing variants into isolated regions including at least a first acidic/acidic region (R1), a second neutral region (R2 ) and a third basic/alkaline region (R3), and determining the percentage of isoforms of charge-different variants falling within each of the R1, R2, and R3 regions.

For such embodiments, image analysis for detection and quantification may be performed according to conventional methods and systems (eg, image analysis software).

In the above embodiments, any of the following additional features/functionality may be included (leading to additional patentable embodiments), however, it should be noted that any one or more of these features may differ and still be within the scope of the present invention - the list below is just one embodiment:

carry out the detection and quantification of different charge options;

detection of different charge variants occurs at a wavelength of approximately 280 nm;

the concentration of protein loaded into the capillary is approximately 2.0 mg/ml;

the stabilizing additive contains urea;

the amount of urea in the mixture is approximately 2M;

the mixture contains approximately 0.35% methylcellulose;

the first voltage is 1500 V;

the first predetermined time is approximately 1 minute;

the second voltage is 3000 V;

the second predetermined time is approximately 7 minutes;

the concentration of protein loaded into the capillary is approximately 1.0 to 8.0 mg/ml;

the amount of urea in the mixture is more than 0 M and less than about 8 M;

the mixture includes from about 0.01 to about 0.35% methylcellulose, or is in any intermediate range;

the first voltage is from about 1 to about 3000 volts, or is in any intermediate range;

the first predetermined time is from about 1 second to about 5 minutes, or any interval in between;

the second voltage is from about 1 to about 3000 volts, or is in any intermediate range; and/or the second predetermined time is from about 1 to about 14 minutes, or is in any intermediate interval.

In some embodiments, an iCIEF capillary configured for use in the assay of charged VEGF-Trap variants is provided and includes a capillary configured for protein production and configured with a mixture of carrier ampholyte, methylcellulose, and a stabilizing additive. The capillary may also contain a fluorocarbon coating.

In some embodiments, an iCIEF kit configured for use in the assay of charge differing VEGF-Trap variants is provided and comprises one or more capillaries configured to produce protein and configured with a mixture of carrier ampholyte, methylcellulose, and a stabilizing additive, wherein the capillary contains a fluorocarbon coating. .

Brief description of graphic materials

Fig. 1A-1F depict VEGF Trap electropherograms using different ampholytes. In FIG. 1A, the ampholyte is a Pharmalyte™ having a pI that is in the range of 3-10; in fig. 1B ampholyte is a combination of Pharmalyte™ with a pI that is in the range of 5-8 and Pharmalyte™ having a pI that is in the range of 8-10.5; in fig. 1C ampholyte represents

- 2 041923 a Servalyt™ having a pI that is in the range of 2-9; in fig. 1D ampholyte is a Servalyt™ having a pI that is in the range of 4-9; in fig. 1E ampholyte is

Biolyte™ having a pI that is in the range of 3-10; and in FIG. 1F ampholyte is a combination of a Pharmalyte™ having a pI that is in the range of 3-10 and a Pharmalyte™ having a pI that is in the range of 6.7-7.

In FIG. 2A-2E show a comparison of VEGF Trap electropherograms at various urea concentrations.

In FIG. 3 shows a comparison of a VEGF Trap electropherogram obtained using isoelectric focusing and capillary isoelectric focusing methods with detection directly in the capillary.

Fig. 4A-4G illustrate analysis of the OFFGEL VEGF Trap fraction (fractions #5-10) using isoelectric focusing and capillary isoelectric focusing methods with direct capillary detection.

Fig. 5A-5F illustrate VEGF Trap RS electropherograms with OFFGEL VEGF Trap fractions as internal standard (fractions #5-10) analyzed in FIG. 4A-4G.

In FIG. 6 shows a comparison of a blank and VEGF Trap electropherogram with an independent marker as an internal standard.

Fig. 7A illustrates a capillary isoelectric focusing electropherogram with detection directly in the capillary of a VEGF Trap RS sample with regions labeled 1, 2 and 3.

Fig. 7B illustrates the difference in electropherogram output between the isoelectric focusing method and the capillary isoelectric focusing method with detection directly in the capillary.

Fig. 8 presents data obtained using capillary isoelectric focusing with detection directly in the capillary compared to the stability of VEGF Trap.

Fig. 9 shows stability data obtained using capillary isoelectric focusing with in-capillary detection performed on forcedly digested VEGF Trap samples.

Fig. 10A-10C illustrate the statistical analysis of capillary isoelectric focusing data with direct capillary detection provided in FIG. 9.

Fig. 11 presents data relating to the linearity of the capillary isoelectric focusing method with detection directly in the capillary.

In FIG. 12 shows a comparison of electropherograms of capillary isoelectric focusing with detection directly in the capillary of three ampholyte samples.

Fig. 13A-13D illustrate statistical analysis of capillary isoelectric focusing data with direct capillary detection obtained from historical VEGF Trap samples.

Detailed description of the invention

This document describes methods and systems for the analysis of different charge variants of different proteins, for example, VEGF Trap. VEGF Trap is a fusion protein containing the sequence shown in table. 1. Instead of outputting a distribution of different charge variants by grouping bands 3-9 in an isoelectric focusing gel, which is the currently approved method for assaying VEGF Trap, the methods and systems described herein typically use capillary isoelectric focusing. with detection directly in the capillary, to measure the charge heterogeneity in percent of isoforms of different charge variants, and group them into three different areas of the electropherogram. This reporting approach may be more sensitive to changes that are observed in isoforms of VEGF Trap samples, as previously mentioned.

Table 1. VEGF Trap sequence

Protein Subsequence SEQID NO VEGF Trap SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLK KFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVN GHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNC TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEM KKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRV HEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 1

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The following abbreviations are used throughout this disclosure:

iCIEF - capillary isoelectric focusing with detection directly in the capillary;

IEF - isoelectric focusing;

pI - isoelectric point;

RS - standard sample;

DS - drug substance;

DSI - drug substance intermediate;

FDS - medicinal substance in the formed mixture.

Methods for assaying charge differing VEGF Trap variants typically involve loading a protein sample into a separating capillary that contains a mixture of at least a carrier ampholyte, methylcellulose, and a stabilizing agent, applying a first voltage for a first predetermined period of time such that the carrier ampholyte forms a gradient pH within the capillary, applying a second voltage for a second predetermined period of time to focus the migration of charge differing protein variants within the capillary such that the total charge of the variants is neutral, and detecting and quantifying the charge differing protein variants.

The separation capillary may be loaded with VEGF Trap at a concentration that is in the range of about 0.5 to about 2 mg/mL.

For example, a separation capillary may 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.

The amount of methylcellulose in the mixture may range from about 0.01% to about 0.35%. For example, the amount of methylcellulose in the mixture may 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%.

As for the first voltage, it may range from about 1 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.

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.

The first predetermined time may range from 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 min (120 s), about 2.5 min (150 s), about 3 min (180 s), about 3.5 min (210 s), about 4 min (240 s), about 4.5 min ( 270 or about 5 minutes (300 seconds) In some embodiments, the first predetermined time is about 1 minute (60.

The second predetermined time may range from about 1 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 min, about 12 min, about 13 min or about 14 min. In some embodiments, the second predetermined time is about 7 minutes.

Any suitable additive may be used in the mixture. In some embodiments, it may be advantageous to use urea as an additive. For example, it may be advantageous to include 2 M urea in the mixture. Various reagents (ampholytes) can also be included in the mixture, as described in more detail below. In one embodiment, VEGF Trap is loaded into a capillary at a concentration of 1.0 mg/ml and analyzed using a capillary isoelectric focusing method with direct capillary detection that uses a mixture of 0.35% methylcellulose, 2 M urea and 3% ampholyte, which has pi 3-10.

Reagents and equipment

Tab. 2 below lists the reagents (ampholytes) and equipment used in accordance with some embodiments of the present disclosure. In the examples, the iCE3 (ProteinSimple®) Charge Differential Variant Analyzer is used. Unless otherwise noted, the standard VEGFTrap (RSVITV-5) was used as the study drug in method development and characterization.

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Table 2. Example of reagents and other components used

Sample Reagent

Pharmalyte™ 3-10

Servalyt™ 4-9

IServalyt™ 2-9

Pharmalyte™ 5-8

Pharmalyte™ 8-10.5

Pharmalyte™ 6.7-7.7

Biolyte™ 3-10 40%

Urea

Methylcellulose

pl marker (5.12, 7.05, 7.65) iCE3 ProteinSimple®

An example of at least some embodiments:

ampholyte screening is initially performed based on the pI range and ampholyte source. Four ampholytes, each covering a unique pI range, are purchased from three different sources. Ampholytes are analyzed using the following input data:

protein concentration 2.0 mg/ml,

M urea,

0.35% methylcellulose, min pre-focus at 1500 V and min focus at 3000 V.

Fig. 1A-F illustrate an electropherogram obtained using six ranges of ampholytes with the iCE3 charge analyzer. The following ampholyte ranges are used:

fig. A - Pharmalyte 3-10, fig. B - combined Pharmalyte 5-8/8-10.5, fig. C - Servalyt 2-9, fig. D - Servalyt 4-9, fig. E - Bio-Lyte 3-10, 40%, fig. F - 3-Blend 3-10 and Pharmalyte 6.7-7.7.

Selected protein

Ampholytes that are in the pI range of 3-10 are chosen as the overall profile of the iCIEF electropherogram, as they most closely resemble the electropherogram from the currently approved procedure for analyzing charge-different VEGF-Trap variants (see, for example, the IEF image shown in Fig. 3).

Urea optimization

Optimization of the method, in accordance with some variants of implementation, also includes changing the concentration of urea (from no urea to 8 M). Fig. 2A-2E illustrate the effect of such a change in urea concentration on the electropherogram of the RSVITV-5 VEGFTrap sample. Although lowering the urea concentration generally improves resolution, it has been found that an increased level of urea (8M) results in a reduction in resolution. However, VEGF-Trap, re-dissolved under natural conditions (without urea), had similar problems with reduced resolution and insufficient reproducibility. Accordingly, the overall peak pattern and resolution were comparable to each other for VEGFTrap when isolated using 1-3 M urea.

Additional experiments were performed using 2 M urea to optimize ampholyte 3-10 concentration and protein concentration. Fig. 3 illustrates electropherograms obtained using 2 M urea, 0.35% methylcellulose and 3% ampholyte 3-10 at a protein concentration of 1.0 mg/ml. The iCIEF electropherogram was compared with another electropherogram and the peaks were prelabeled (FIG. 3).

Method characteristic

The total charge profile and peak pattern obtained using the iCIEF analysis method were consistent with the IEF band profile (as shown in FIGS. 2A-2E). However, in order to further understand and correlate the band pattern of the IEF assay method with the pattern of peaks obtained using the iCIEF assay, an OFFGEL fractionation of the VEGF-Trap sample was performed. Fractions of individual charge different variants obtained from OFFGEL electrophoresis are analyzed using IEF and iCIEF analysis methods. An Agilent 3100 OFFGEL fractionator is used to fractionate a VEGF-Trap standard with its isoelectric points, and individual isoforms recovered as liquid fractions are analyzed using two methods, IEF and iCIEF. VEGF-Trap RS is fractionated using fixed pH gradient strips (IPG strips) at pH 6-9, which covers the pI range of the charge differing variants observed for VEGF-Trap. The detailed scheme of the experiment and separation is as follows:

- 5 041923

1) stock solution is prepared by combining 2.3 ml 50% glycerol, 230.4 µl IPG buffer (pH

6-11) and 16.64 ml of water for a total volume of 19.2 ml;

2) VEGF Trap solution is prepared by adding 73 μg VEGF (4.2 mg) to 12 ml stock solution and 3 ml water;

3) IPG strip rehydration solution is prepared in excess by combining 1.15 ml of water with 4.6 ml of stock solution;

4) IPG gel strips, pH range 6-9 (24 cm), are placed on each other lane of two instrument trays and 24 well frames are fixed above them. For strip rehydration, loading of antibodies, loading of trays on the instrument, the standard OFFGEL kit protocol is used (see OFFGEL user manual: Agilent 3100 OFF GEL Fractionator Kit quick Start Guide, 5th Edition Sep 2010);

5) use a platform temperature of 20°C. A standard protein focusing instrument for a 24-well setup was run using 50 µA DC, 8000 V maximum voltage setting, and 200 mW maximum power setting;

6) after 34 hours of fractionation, the run is stopped and similar numbers of wells from each lane for wells 3 to 12 are pooled and then replaced with water and concentrated approximately 5 times before analysis;

7) the amount of antibodies in each fraction is determined by measuring the absorbance on Nanodrop at 280 nm with an extinction coefficient of 1.15. One tool is to determine the concentration of a fraction and then multiply the volume of the fraction by the concentration.

Briefly, 4.2 mg RS VEGF-Trap was fractionated using 12 IPG strips over 32 hours; individual fractions from each strip corresponding to the same pI range are pooled and quantified after dialysis. After fractionation, a total of seven fractions (fractions 4-10) that had sufficient recovery were analyzed using IEF and iCIEF. Fractions are analyzed in two ways: Separate analysis of OFFGEL fractions (4-10) using IEF and iCIEF analysis methods (see Fig. 4) and adding OFFGEL fractions (5-10) as an internal standard in RS VEGF-Trap followed by analysis internal standard samples using IEF and iCIEF (FIG. 5). Tab. 3 shows the fractions and corresponding amounts obtained in the OFFGEL fractionation study.

Table 3

Fraction OFFGEL No. Conc, protein (mg/ml) Received volume (µl) 3* 1.229 15 4 1.628 98 5 1.884 104 6 2.909 FROM 7 3.498 124 8 3.56 124 9 3.326 124 10 2.098 110 AND* 1.235 94 12* 2.574 15

In addition to analyzing the OFFGEL fractions independently using IEF and iCIEF, the fractions (5-10) that gave higher recovery had a 1:0.1 internal standard (RS VEGF-Trap:fraction) and were analyzed with two ways to analyze variants that differ in charge. Fig. 5 illustrates a panel of electrophoregrams with VEGF-Trap RS as internal standard with OFFGEL (5-10) VEGF-Trap fractions analyzed using iCIEF (upper panel) and IEF (lower panel) assay methods. For each of the OFFGEL fractions as internal standard in RS, the corresponding control RS (without internal standard) is superimposed on the IEF and iCIEF assay methods. Overlapping samples with and without internal standard (RS+OFFGEL and RS) aids in visualizing and understanding the correlation of peak patterns between IEF and iCIEF assays. In each panel, the amplification of the charge species corresponding to the internal standard fraction is indicated by an arrow ranging from acidic fractions (FIG. 5A) to basic fractions (FIG. 5F).

The correlation between the gel-based IEF band pattern and the capillary-based iCIEF peak pattern was evident from the analysis of the OFFGEL fractions. From FIG. 4 and 5 (analysis of single fractions with internal standard fractions), it can be concluded that the charge isoform separation achieved by the currently approved IEF gel method is comparable and similar to the peak pattern obtained using the method on iCIEF capillary base for VEGFTrap.

Issuance of results on the distribution of variants differing in charge using the iCIEF method. The currently approved IEF method for distributing charge-different VEGF-Trap variants indicates percent charge variance by clustering bands 3-9 on an IEF gel. Interest

- 6 041923 areas of bands 3-9 are summarized and reported using, for example, myoglobin (an independent protein marker) as a guide to determine the number of bands based on myoglobin pI. The SPEC acceptance criterion for the IEF method is 82% (lanes 3-9).

A similar approach was adopted for the new iCIEF assay method, where the independent marker from ProteinSimple, pI 7.05 Marker cat. No. 102226 is added to the iCIEF master mix as an internal standard (2 M urea, 0.35% methylcellulose, 3% ampholyte 3-10). The electropherogram of iCIEF with marker 7.05 as an internal standard in the master mix is shown in FIG. 6 (top panel), the limit markers pI 5.12 and pI 9.50 in the master mix are used for calibration. The bottom panel in Fig. 5 illustrates an iCIEF RS VEGF-Trap electropherogram superimposed on a blank sample containing marker 7.05 as an internal standard that will be used to identify Peak 5 in the iCIEF VEGF-Trap sample profile. Peak 7.05 of the marker migrates at pI between peaks 4 and 5 and this will serve to identify major peak 6 in the cluster of major VEGF-Trap isoforms (Peaks 5, 6 and 7).

Instead of showing peaks 3-9 as in the IEF method, the Icief method (according to some embodiments) demonstrates charge heterogeneity of the VEGF-Trap sample in percent isoforms of different charge variants grouped into region 1 (acidic), region 2 ( neutral) and region 3 (main). A cluster of three major peaks (peak numbers 5, 6 and 7) on the iCIEF VEGF-Trap electropherogram that migrate around the neutral pI range and that correspond to the most prominent isoforms will be grouped as region 2 (neutral). Among the cluster of three peaks, a distinct isoform corresponding to major peak 5 that migrates to a particular pI was identified using the independent marker pI 7.05 as an internal standard in a blank injection as shown in FIG. 7. Region 1 (acidic) in the iCIEF VEGFTrap sample is indicated as a group of peaks that are relatively acidic compared to the cluster of three main peaks (Peaks 5, 6 and 7) in the VEGF-Trap electropherogram. Region 3 (main) in the iCIEF VEGF-Trap sample is indicated as a group of peaks that are relatively basic compared to the cluster of three main peaks (Peaks 5, 6 and 7) in the VEGF-Trap electropherogram.

The region 1, 2, and 3 reporting approach has the advantage of tighter control of isoforms of charge-different variants by monitoring three regions (Regions 1, 2, and 3) as opposed to the traditional grouping of lanes 3-9 of the gel-based IEF method. Fig. 7B illustrates the differences between the output of IEF results and the output of results in accordance with embodiments of the iCIEF method of the present disclosure. In addition, reporting results for regions 1, 2, and 3 gives the iCIEF assay method the unique advantage of being sensitive to changes in isoforms of charge-different variants much earlier than the traditional approach. This makes the iCIEF assay much more sensitive in its readings and a better indicator of stability than the previous IEF assay procedure. In table. 4 below is an example of stability, indicating the ability of the new grouping approach adopted for the iCIEF analysis method as opposed to the traditional output of IEF analysis results 3-9. VEGF-Trap samples used in accelerated stability testing are analyzed using the new iCIEF assay using two reporting approaches - region and peak 3-9 grouping similar to the IEF assay method and compared to historical results from the IEF assay method.

In table. 4 (below) it can be seen that the grouping approach to regions 1, 2 and 3 is much more sensitive and indicative of changes in the distribution of different charge variants of the VEGF-Trap sample. The IEF method showed a change in the total charge distribution with a 2% decrease in the case of bands 3-9 and this change was comparable to the results of the iCIEF analysis method when grouping using the approach for peaks 3-9. However, from Table. 4 shows that for the VEGF-Trap sample used in the accelerated stress test at 25°C, a 5% increase in region 1 (or acid variants) and a concomitant decrease of approximately 5% in region 3 (basic variants) was observed using the assay iCIEF. This trend observed in the charge distribution of VEGF-Trap in the iCIEF assay is an accurate reflection of the nature of the changes occurring in the VEGF-Trap sample based on its structure and the complexity of the charge pattern due to its varying degrees of sialylation. The increase in acid variants (Region 1 - high degree of steel particles) of the VEGF-Trap sample using the iCIEF assay method in accelerated thermal stress tests reflects possible deamidation in combination with aggregation. On the other hand, clustering using traditional bands 3-9 with the IEF method masks the subtle changes occurring in the different charge isoforms of VEGF-Trap and leaves little room for monitoring different charges, making it not as sensitive as a way to detect subtle changes in charge heterogeneity of the VEGF-Trap sample.

VEGF-Trap has ten glycosylation sites. The glycan chains attached to these sites are branched, and each branch may or may not terminate in a negatively charged sugar monomer, sialic acid. Natural variation in the presence of sialic acid groups at the ends of glycan chains results in an ensemble of charge-different variants

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VEGF-Trap having a full charge range. The proportion of these bands varies depending on the abundance of charged species present. Thus, a novel approach to reporting grouping different kinds of charges based on areas 1 (highly steeled), 2 (moderately steeled) and 3 (least steeled) makes the iCIEF analysis more sensitive to changes that occur in the sialyl forms of the VEGF-Trap sample. .

Table 4

Stress condition iCIEF IEF iCIEF %R1 %R2 %R3 % bands 3-9 % peaks 3-9 VEGF 25°С 1 month 31.90 43.09 25.01 82.94 87.48 VEGF 25°С 3 months 33.85 43.28 22.87 82.11 87.86 VEGF 25°С 6 months 37.25 42.47 20.28 80.42 85.15 Difference (%) 5.35 -0.62 -4.73 -2.52 -2.33

Stability, indicating the ability of the iCIEF assay method

Sample DP VEGF-Trap for long-term stability testing (kept at a temperature of 2-8°C) analyzed using 7 independent time points covering a time period of 24 months; tab. 5 (below) illustrates data relevant to this study. Historical IEF data for these VEGF-Trap samples are offered for comparison and compared with iCIEF VEGF-Trap data from region grouping and peak 3-9 results. Under real-time storage conditions of 2-8°C, VEGF-Trap samples show little or no change based on historical IEF data, a similar trend is observed when reporting results using the iCIEF approach to peaks 3-9.

Table 5. VEGF-Trap sample analyzed using iCIEF _____________ in real time__________

Condition Time %R1 %R2 %R3 IEF (%39) iCIEF (%3-9) 5°C 3 months 33.67 46.49 19.84 89.21 85.45 5°C 6 months 32.90 47.14 19.96 87.09 85.26 5°C 9 months 33.20 46.99 19.81 88.78 85.23 5°C 12 months 33.71 47.05 19.24 91.37 85.12 5°C 15 months 33.71 46.80 19.49 89.92 85.05 5°C 18 months 33.89 47.00 19.11 88.27 85.25 5°C 24 months 34.14 46.87 18.99 89.79 85.03

Fig. 8 illustrates the fitting of the direct % of VEGF-Trap regions 1, 2 and 3 over a 24 month time period using iCIEF. A small but steady increase in area 1 with a decrease in area 3 can be seen in the graphs.

An additional analysis using a forcedly digested DS VEGF-Trap sample was performed using the new iCIEF assay method. For this study, a thermally decomposed sample of DS VEGF that was diluted and stressed at 45°C for a period of 15 days was analyzed at time points 0, 3, 9 and 15 days using the iCIEF method (by region and 3-9) . From Table. 6 (below) shows that a slight increase in acid charge variants (region 1) is observed for the iCIEF assay method when grouped using the region approach compared to reporting peaks 3-9 at a much earlier point in time for the force-degraded VEGF sample. -Trap. While the output for % 3-9 shows a 2% change in total charge distribution over peaks 3-9, region 1 under the same conditions showed a 7% increase while region 3 showed a concomitant 8% decrease over time . Fig. 9 illustrates an electropherogram of a forcedly digested VEGF-Trap sample using the iCIEF analysis method; an increase in acidic species can be seen from the profile.

Table 6. Percent Distribution of Forced Degraded VEGF-Trap Sample Analyzed Using iCIEF (Thermal Stress)

Stress condition iCIEF iCIEF %R1 %R2 %R3 % peaks 3-9 DS VEGF 45°C Day 0 28.66 44.30 27.04 84.54 DS VEGF 45°C Day 3 29.47 44.60 25.93 83.90 DS VEGF 45°C Day 9 31.83 45.88 22.29 83.56 DS VEGF 45°C Day 15 35.65 45.35 19.00 82.44 Difference (%) 6.99 1.05 -8.04 -2.10

Statistical analysis of the % Distribution of the three regions for the stressed VEGF-Trap sample is performed by comparison with the corresponding percentage of peaks for non

-8041923 stressed VEGF sample. In FIG. 10A-10C show statistical analysis of a stressed VEGF-Trap sample for iCIEF data. A statistically significant change is observed for regions 1 and 3, respectively.

Based on the characterization and optimization of the iCIEF analysis method, the analysis parameters are obtained and presented in Table. 7 (below). These method conditions are evaluated for linearity, accuracy, precision, and within-laboratory error.

Table 7. Critical Method Parameters Obtained from ____________________ Development and Optimization Experiments________________

Urea 2M Methylcellulose 0.35% ampholyte 3-10 3% VEGF-Trap 1.0 mg/ml Pre-focusing 1 min. at 1500 V Focusing 7 min. at 3000 V

iCIEF analysis method validation. Linearity of the iCIEF analysis method. The linearity of the method is evaluated by one analyst. Sample solutions were prepared using the VEGF-Trap standard at various protein concentrations of 0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, and 2.0 mg/mL. In this experiment, the protein concentration in the sample matrix is changed from 0.5 to 2 mg/ml, keeping other components of the matrix constant at 3% ampholyte 3-10 and 0.35% methylcellulose. The focusing time also remains constant at 1+7 minutes. In table. 8 summarizes the percent distribution of regions 1, 2, and 3 for the VEGF-Trap sample over a linear range of 0.5 to 2.0 mg/mL. A line plot of concentration versus area ratios for a VEGF-Trap sample is provided in FIG. eleven.

The assay shows acceptable linearity over the protein concentration range of 0.5 to 2.0 mg/mL with R2 > 0.99 based on regression analysis. Moreover, the isoform distribution remains unchanged over the same concentration range. This indicates that the assay is able to provide consistent results in both peak area and isoform distribution over the protein concentration range of 0.5 to 2.0 mg/mL.

Accuracy of the iCIEF analysis method

The accuracy of the method is evaluated based on dilution proportionality using linearity data by comparison with a nominal concentration of 1.0 mg/mL. Dilution recovery based on linearity data is shown in Table 1. 9 below. The percentage recovery is calculated using = (percentage of measurement area/percentage of nominal area) x 100%.

Table 9. Accuracy of the iCIEF VEGF-Trap Assay Based on Dilution Proportionality

Average % % Recovery

RS VEGF-Trap (mg/ml) %R1 %R2 %R3 %R1 %R2 %R3 0.5 29.65 46.41 23.95 99.35 101.09 98.78 1 29.85 45.91 24.24 Rated 1.5 29.98 45.89 24.14 100.44 99.96 99.59 2 29.56 46.29 24.16 99.03 100.82 99.67

Restoration based on proportionality when diluted in the protein concentration range from 0.5 to 2.0 mg/ml for the VEGF-Trap sample was in the range of 98-101% for the three regions.

Analysis of the intralaboratory error of the iCIEF method. Within-laboratory error was assessed by two different analysts (A, B) using preparations based on their respective reagents and using two iCE3 different charge variants analysis devices at four days for a VEGF-Trap RS sample. The results of the analysis are given in table. 10 below.

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Table 10. Results of the analysis of intralaboratory error

Analyst % area 1 % area 2 % area 3 A 29.44 45.87 24.69 30.53 45.08 24.39 29.71 45.83 24.46 A 29.91 45.98 24.11 29.50 46.18 24.31 29.76 46.14 24.10 IN 28.78 46.65 24.57 29.40 46.22 24.38 29.61 45.85 24.54 IN 29.56 46.10 24.34 30.01 45.85 24.15 29.50 45.89 24.61 General average 29.64 45.97 24.39 Art. off 0.42 0.37 0.20 % RSD 1.40 0.80 0.81

The proposed iCIEF VEGF-Trap assay demonstrates acceptable precision when performed by different analysts using different reagent preparations. Calculate the total RSD (relative standard deviation) in % and it is within the RSD of 2% for all three areas.

Robustness of the iCIEF analysis method. Several elements of the robustness of the analysis method were evaluated using various experiments. These experiments are shown in table. eleven.

Table 11. Summary of experiments on the robustness of the method

Robustness experiments Performance evaluation The stability of the prepared solution Constancy of isoform distribution over 24 hours compared to T=O time Evaluation of other ampholyte samples 3-10 Constancy in the overall profile and % distribution across the three areas Stability indication data Effect of stress on isoform distribution

The stability of the prepared solution in the device. Solution stability is assessed by obtaining a sample from a standard sample in a sample matrix and analyzing the image using iCIEF. The sample is stored in the iCE3 instrument after analysis at 10°C in a matrix consisting of 3% ampholyte 3-10, 0.35% methylcellulose and 2 M urea. The sample is analyzed again after approximately 24 hours. The distribution of the isoform for each analysis is presented below in table. 12 below.

Table 12. Stability results of the prepared iCIEF sample in the device

Sample name % area 1 % area 2 % area 3 injection of 1 RS VEGF at time 0 29.49 45.72 24.80 injection of 2 RS VEGF at time 0 29.88 45.62 24.50 injection of 1 RS VEGF at time point 24 hours 29.52 45.92 24.56 injection of 2 RS VEGF at time point 24 hours 29.95 45.74 24.31 % difference 0.46 0.30 0.49

The absolute difference between a sample analyzed at time T=0 and analyzed again at time T=24 h is calculated based on the range (Maximum and Minimum) observed at each time point. The absolute difference was equal to or less than 0.5% for the three regions. This indicates that the sample is stable in the matrix for up to 24 h when stored at 10°C in the analyzer of different iCE3 variants.

Evaluation of ampholyte samples (3-10). Analyzed several samples of ampholyte 3-10 from one source. The overall profile of the different charge variants of the VEGF-Trap sample analyzed using different samples was comparable. Slight differences in the electropherogram profile in terms of peak pattern were observed in region 1 for some ampholyte samples, however, the distribution percentage was similar and within the range of analysis results. Representative electropherograms from three ampholite samples are shown in FIG. 12.

Example. Analysis of Historical VEGF-Trap Variant Samples (DS, FDS and DSI) Using the New iCIEF Analysis Method

In order to further establish the robustness of the iCIEF assay method, using iCIEF, a total of 37 unique historical VEGF-Trap samples were analyzed using two ampholyte samples that are unrelated in origin. The assay included 15 DSI VEGF-Traps (substance intermediate, aqueous buffer, pH 6.2, containing 5 mM sodium phosphate, 5 mM sodium citrate, and 100 mM sodium chloride), 10 DS VEGF-Traps (drug substance C701 SPEC, aqueous buffer solution, pH 6.2, containing 10 mM sodium phosphate) and 12 samples of FDS VEGF-Trap (drug substance in the formed mixture, aqueous buffer solution C713 SPEC, pH 6.2, containing 10 mM sodium phosphate, 40 mM chloride sodium, 0.03% (w/v) polysorbate 20 and 5% (w/v) sucrose). These VEGF-Trap samples were analyzed using the iCIEF analysis method.

Ampholytes are a mixture of different homologues of amphoteric compounds with a spectrum of isoelectric points between 3 and 10, which help to set the pH gradient under the influence of an electric field. Ampholyte 3-10 used in the iCIEF assay method was purchased from a single source, which is usually produced in batches. Based on the supplier's recommendation, together with our working knowledge of iCIEF assays for other proteins, small differences between different samples are observed and are unavoidable. Therefore, to establish the robustness of the new iCIEF assay in different ampholyte samples, two ampholyte samples were analyzed. VEGF samples from the DS, DSI and FDS product step were analyzed using the proposed region clustering approach (R1, R2 and R3) and based on peak clustering 3-9 similar to the IEF analysis method. Fig. 13A-13C show statistical analysis of ampholyte samples as a function of % region 1, 2, and 3 and peaks 3-9 for VEGF-Trap DS, DSI, and FDS samples. Data corresponding to 37 VEGF-Trap samples are provided in Table 1. 13 for one of the ampholyte samples. Tab. 14-16 offer a complete set of data for 37 samples.

Table 13 Historical DSI, DS and FDS VEGF-Trap samples were analyzed using the iCIEF analysis procedure.

Sample DSI VEGF Trap %R1 %R2 %R3 % 3 to 9 peaks 1 29.7 43.6 26.7 82.7 2 28.5 43.6 28.0 83.5 3 28.0 44.3 27.7 84.2 4 28.1 44.2 27.8 83.9 5 28.7 44.9 26.4 84.6 6 27.6 44.2 28.2 84.0 7 23.7 41.8 34.6 81.7 8 26.8 43.1 30.1 82.9 9 28.5 45.2 26.4 85.0 10 27.2 44.1 28.7 84.0 AND 28.5 45.2 26.4 85.0 12 27.9 44.3 27.8 84.4 13 28.0 43.7 28.3 83.7 14 27.8 42.9 29.3 83.3 15 29.2 44.7 26.1 84.3 16 29.0 44.0 27.0 84.2 17 28.9 43.7 27.4 83.8

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18 30.6 44.3 25.1 84.1 19 29.7 43.9 26.4 83.8 20 29.9 43.7 26.4 83.3 21 29.1 43.9 27.1 84.1 22 27.1 44.8 28.1 85.3 23 30.4 44.7 24.9 84.8 24 28.3 43.7 28.1 83.2 25 27.3 43.3 29.5 83.2 26 28.0 42.3 29.6 81.8 27 27.2 45.2 27.6 85.8 28 30.1 43.4 26.5 84.1 29 29.2 44.1 26.7 84.0 thirty 29.8 43.4 26.8 83.3 31 29.7 44.7 25.7 84.6 32 32.3 45.3 22.4 84.5 33 33.4 44.4 22.2 83.8 34 33.6 45.2 21.1 84.5 35 31.0 45.2 23.8 85.3 36 33.8 42.1 24.1 81.3 37 29.2 42.2 28.6 82.2 38 28.2 41.9 29.9 81.8 39 28.7 42.9 28.4 82.8

Table 14. DSI Lots Assayed from Pharmalyte Samples

DSI VEGF Trap Samples %R1 %R2 %R3 % 3 to 9 peaks 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 eleven 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

Table 15. DS Lots Assayed from Pharmalyte Samples

Samples of DS VEGF Trap %R1 %R2 %R3 % 3 to 9 peaks 1 28.8 44.7 26.4 89.3 2 29.4 44.3 26.3 89.2 3 29.2 44.5 26.2 88.9 4 27.3 43.4 29.2 88.2 5 29.6 44.0 26.4 88.9 6 28.3 43.8 27.9 88.8 7 26.7 45.3 28.1 89.8 8 30.2 45.1 24.7 90.1 9 30.5 44.5 25.0 89.3 10 29.1 43.9 27.0 89.3 AND 27.9 42.5 29.5 87.5

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Table 16. FDS Lots Assayed from Pharmalyte Samples

FDS VEGF Trap Samples %R1 %R2 %R3 % peaks since 3 by 9 1 27.5 45.0 27.5 90.0 2 30.0 43.5 26.5 88.7 3 29.1 43.7 27.2 89.1 4 30.2 43.0 26.7 88.4 5 29.8 44.6 25.6 89.8 6 32.5 45.4 22.1 90.3 7 33.3 44.7 22.0 89.6 8 33.6 45.4 21.0 90.9 9 30.7 45.5 23.8 90.1 10 34.2 41.7 24.2 87.2 AND 29.0 42.4 28.6 87.2 12 28.4 41.3 30.3 86.5 13 28.5 42.5 29.1 87.6

Matched pair analysis (FIG. 13D) was performed based on the data collected for the ampholyte samples for region 1,2 and 3 and the grouping approach 3-9. Data analysis from matched pairs was used to compare the means between the two ampholytes and to assess any difference in the output of the analysis results that may be due to internal differences in the ampholyte samples. Based on the data, it can be concluded that the maximum observed average difference between samples for the three regions RI, R2 and R3 is less than 0.1% if reported in the case of regions. In addition, based on the p-value, it can be concluded that the % distribution in area 1, 2, and 3 between the two ampholyte samples using the area approach is not statistically significant.

However, when the VEGF-Trap data set for DS, DSI, and FDS samples was analyzed using a 3-9 peak clustering approach similar to the IEF analysis method, there was a statistically significant difference between some ampholyte samples. For example, between some samples, the average difference is as high as 4.8% if indicated in the case of peaks 3-9 for the iCIEF assay method. Fig. 13A-13D show iCIEF profiles obtained using various ampholyte samples, and from the images it can be seen that the variability observed between ampholyte samples is limited to the acidic region and by grouping regions 1, 2 and 3 this variability is masked. This makes the area approach to reporting results for iCIEF analysis a robust and reproducible approach.

Data from DSI, DS, and FDS sample analysis using ampholyte samples, based on grouping % of regions 1, 2, and 3, makes iCIEF analysis a more robust analysis method.

Accordingly, the iCIEF system, methods, and devices provided in this disclosure quantify the profile of charge-different VEGF-Trap drug substance variants, drug substance intermediate, drug substance in the formulated mixture, and drug product. Such embodiments may serve to replace the currently approved gel-based IEF method for VEGF-Trap charge heterogeneity analysis.

Charged variants of VEGF-Trap fractionated using the OFFGEL 3100 fractionator demonstrates the correlation between the peaks obtained in the capillary-based iCIEF assay method with the bands resolved in the gel-based IEF assay procedure. By analyzing the VEGF-Trap fractions in OFFEGEL electrophoresis individually and by using jumps in the assays, a direct comparison of individual iCIEF peaks with the bands of charge-different VEGF-Trap IEF variants is achieved. Studies have confirmed that the new iCIEF assay procedure is able to separate all isoforms of charge-different variants previously separated using the gel-based IEF method with an equivalent and more accurate method.

Accordingly, some embodiments of this approach to results based on the percentage distribution of regions 1,2 and 3 disclosed herein allows control of all VEGF-Trap isoforms and makes the assay more sensitive, allowing it to be a robust stability assay.

Capillaries for use with iCEF methods are also described in this document. In general, an iCIEF capillary configured for use in the assay of different charge VEGFTrap variants, and it includes a capillary configured for protein production and configured with a mixture of carrier ampholyte, methylcellulose and a stabilizing additive. The capillary may also contain a fluorocarbon coating.

In some embodiments, an iCIEF kit configured for use in the assay of charge differing VEGF-Trap variants is provided and comprises one or more capillaries configured to produce protein and configured with a mixture of carrier ampholyte, methylcellulose, and a stabilizing additive, wherein the capillary contains a fluorocarbon coating. .

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Claims (8)

Although various patentable embodiments have been described and illustrated herein, many other methods and/or structures for providing a function and/or obtaining results and/or one or more benefits will readily be identified by those skilled in the art. described herein, and each of such variations and/or modifications is considered to be within the scope of the embodiments of the invention described herein. In general, those skilled in the art will readily appreciate that all parameters, amounts, percentages, concentrations, sizes, materials, and configurations described herein imply that the actual parameters, amounts, percentages, concentrations, sizes, materials, and/or configurations will depend on the particular application or applications for which patentable ideas are used. One of ordinary skill in the art will understand or be able to determine many equivalents to the specific embodiments of the invention described herein using no more than routine experimentation. Therefore, it should be understood that the foregoing options are provided by way of example only. Patentable embodiments disclosed herein may be practiced in a manner other than that described and claimed. Patentable embodiments of the present disclosure also include the individual features, system, formulation, material, kit, and methods described herein. In addition, any combination of two or more such features, systems, formulations, materials, kits, and methods is also patentable (unless they are mutually incompatible). Some embodiments may differ from the prior art in that, in particular, one or more features/elements/functionality are missing (ie, claims directed at such embodiments may include negative limitations). In addition, as noted, various ideas of the invention can be embodied in one or more ways. Actions performed as part of a method may be performed in any suitable manner. Accordingly, embodiments may be constructed in which the actions are performed in a different order from that illustrated, which may include performing some of the actions at the same time, even though they are shown as sequential actions in the illustrative embodiments. Any and all references to publications or other documents provided anywhere in this application are incorporated herein by reference in their entirety. In addition, it should be understood that all definitions as defined and used herein control dictionary definitions, definitions in documents incorporated by reference, and/or the conventional meanings of certain terms. CLAIM 1. Capillary Isoelectric Focusing with Direct Capillary Detection (iCIEF) method for analyzing charge differing variants of the vascular endothelial growth factor (VEGF) protein inhibitor VEGF-Trap, which comprises loading a protein sample into a separating capillary that contains a mixture of at least , carrier ampholyte, 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 charge-different VEGF-Trap protein variants within the capillary such that the overall charge of the variants is neutral; and detection and quantification of different charge variants of the VEGF-Trap protein, including: (i) measuring the uptake of multiple isoforms of different charge variants; (ii) separating the plurality of isoforms of charge-different variants into isolated regions including at least a first acidic/acidic region (R1), a second neutral region (R2) containing three major peaks, and a third major/alkaline region (R3); and (iii) determining the percentage of isoforms of charge-different variants falling within each of the R1, R2, and R3 regions. 2. The method according to claim 1, where the detection of different charge variants occurs at a wavelength of 280 nm. 3. The method according to claim 1, wherein the concentration of the protein loaded into the capillary is 2.0 mg/ml. 4. The method according to claim 1, where the amount of urea in the mixture is 2 M. 5. The method according to claim 1, where the mixture contains 0.35% methylcellulose. 6. The method according to claim 1, where the first voltage is 1500 V. 7. The method of claim 1, wherein the first predetermined time is 1 minute. 8. The method according to claim 1, where the second voltage is 3000 V. -