IgG Purity/Heterogeneity and SDS-MW Assays with High- Speed Separation Method and High Throughput Tray Setup High Throughput Methods to Maximize the Use of the PA 800 Plus system Jose-Luis Gallegos-Perez SCIEX Separations, Framingham, MA Introduction The PA 800 Plus Pharmaceutical System provides a comprehensive, automated, and quantitative solution for the characterization and analysis of therapeutic proteins. The application menu for the PA 800 Plus includes, among others, the IgG Purity/Heterogeneity assay and the SDS-gel molecular weight (SDS-MW) analysis.,2 The first method allows for the resolution of reduced and non-reduced immunoglobulins by size and to subsequently quantify the heterogeneity and impurities that may be present in IgG preparations. The SDS-MW analysis is designed to provide an estimation of the molecular weight of a protein in a sample. There are two types of analysis methods that can be used with these assays: ) The high resolution (HR) method with an inlet to detection window capillary length of 20.0 cm, using the capillary cartridge in a left-to-right configuration. 2) The high-speed (HS) method that uses the capillary cartridge in the right-to-left configuration with a sample introduction inlet to detection window capillary length of 0.0 cm. Both methods are used with a tray configuration that allows running up to 2 samples before having to change reagents. However, other tray setups are possible to optimize and maximize the number of sample runs. This report illustrates the use of the HS method and proposes a High Throughput Tray (HTT) setup to maximize the number of samples per run. We demonstrate that HS and HR analysis methods can be used with this optimized tray array. Advantages of the HS Analysis Method with the High Throughput Tray Setup The HS method provides faster separation time (-20 min vs. 0 min of the HR method); it represents 72 samples/day instead of 8/day. PA 800 Plus system The use of an HTT setup allows for analysis of up to 8 samples (2 using the conventional tray array) before changing chemical reagents. It represents savings in consumables and time. Using the HS method and high throughput tray setup saves time and reagents and helps maximize the use of the PA 800 Plus. Experimental Procedure Reagents: Separations using the IgG Purity Heterogeneity Assay Kit and SDS-MW Kit were performed as described in their respective manuals.,2 Iodoacetamide (IAA) and 2- mercaptoethanol were purchased from Sigma-Aldrich and used without any further purification. Ultrapure water (type ) was obtained from a Milli-Q Direct Ultrapure Water System. Bovine Serum Albumin (BSA) and Myoglobin (Myo) were purchased from Sigma-Aldrich. Capillary: A bare fused-silica capillary was used for separation of monoclonal antibody samples; 0 m i.d. x 0 m o.d. x 0.2 cm total length. Inlet-to-detection window equal to 20 cm for the HR method and 0 cm for the HS method. p
Sample Preparation: IgG control standard: A vial containing 9 L of the IgG control standard was combined with 2 L of the 0 kda internal standard and L of 2-mercaptoethanol in a fume hood. The vial was mixed thoroughly and centrifuged at 00g for min. The vial was sealed with Parafilm and heated to 70 C for 0 minutes, after which the vial was cooled for minutes in a room-temperature water bath. Non-reduced IgG sample preparation: A vial containing 9 L of the IgG control was combined with 2 L of the 0 kda internal standard and L of a 20 mm IAA solution. The vial was mixed thoroughly and centrifuged at 00g for minute. The vial was sealed with Parafilm and heated to 70 C for 0 minutes, after which the vial was cooled for minutes in a room-temperature water bath. HTT configuration: HR method: Figure 2 shows the position of the buffer vials in each inlet and outlet buffer tray using the HTT setup. In this configuration the buffer vials in each row are enough for 8 consecutive experiments, therefore it is possible to run up to 8 individual samples. (8 runs per row x rows). Method parameters are shown in Figure. SDS-MW Size Standard: 0 L of molecular weight size standard, 8 L of sample buffer, 2 L of internal standard, and L of 2-mercaptoethanol were combined in a vial. The mixture was heated to 00 C for minutes and placed into a room-temperature water bath to cool down for five minutes before injection. BSA and Myo were diluted with the SDS-MW sample buffer to a final concentration of 0. mg/ml each. For non-reduced protein preparation, a vial containing 9 L of sample solution was combined with 2 L of 0 kda internal standard and L of 20 mm IAA solution. The vial was mixed thoroughly and centrifuged at 00g for minute. The vial was sealed with Parafilm and heated to 70 C for min. For the reduced protein sample, 9 L of the sample was combined with 2 L of the 0 kda internal standard, and L of 2- mercaptoethanol. The mixture was heated to 00 C for min. HS method with conventional tray setups Because the HS method uses the capillary cartridge in the rightto-left configuration, the buffer tray on the right must be used as the inlet tray while the buffer tray on the left must be used as the outlet tray (see Figure ). The polarity for injection and separation should be normal. Using the conventional tray configuration, a maximum of 2 individual samples can be analyzed without reloading the trays with fresh solutions. Outlet Inlet Figure : Image of the inlet and outlet buffer vial tray set-up for the HS method in the PA800 Plus. Gel-R Gel-S NaOH HCl Gel-R Gel-S NaOH HCl Gel-R Gel-S NaOH HCl Gel-R Gel-S NaOH HCl 2 Gel-R Gel-S NaOH HCl Gel-R Gel-S NaOH HCl INLET BUFFER TRAY Method HR HS* Tray on the left Tray on the right 2 OUTLET BUFFER TRAY * Use only rows - (see text) Figure 2: Scheme of the inlet and outlet buffer vial tray setup for the HTT configuration using the HR or the HS method. Ensure the position of the buffer trays in the instrument according to the method being used. (HS method for no more than 0 individual samples, see text). Gel-S = Gel used for separation, Gel-R = Gel used for rinsing, both are of the same gel type. p 2
HS method: If less than 0 individual samples are being analyzed, buffer trays must be set up as shown in Figure 2 using rows from to only. IMPORTANT: In this case, the F-column in the SAMPLE TRAY must be kept empty, with no samples to avoid a tray collision during analysis. Follow the method parameters shown in Figure. HS method and HTT setup to maximize the number of sample analyses: To use the HTT configuration with the HS method for analysis of up to 8 samples requires switching reagents in positions C and D in the outlet and inlet buffer trays to avoid tray collision when samples are injected (see Figure ). All columns in the sample tray (from A to F) can be loaded with samples. For this specific case, use the method parameters show in Figure. 8 7 2 8 7 Sample Tray Sample Gel-R NaOH 2Gel-S HCl Sample Table indicates the volume of reagents used in each buffer vial and their number for all 8 runs using the HTT setup. Table. Volume in Buffer Vials. Reagents Vol (ml) Number of vials (8 runs) Water. 2 Gel. Water. 8 Waste (water) 0.8 2 NaOH. HCl. Instrument: Separations were performed using a PA 800 Plus Pharmaceutical Analysis System from SCIEX. PDA detector settings were configured according to the application guides. The instrument was controlled using 2 Karat software v0. The sample and capillary storage temperatures were set at ºC and 2ºC respectively. CE Instrument setups for the HR and HS methods with the HTT configuration are shown in Figures, and. Waste Waste HGel-S 2 O Waste Waste Waste Waste Gel-S Waste Waste Waste Waste Gel-S Waste Waste Waste Waste Gel-S Waste Waste 2 Waste Waste Gel-S Waste Waste Waste Waste Gel-S Waste Waste 2 Waste Gel-R NaOH Gel-S HCl Waste Waste Gel-S Waste Outlet Buffer Tray Inlet Buffer Tray Figure : Scheme of the inlet and outlet buffer vial tray setup for the HTT configuration using the HS method to inject up to 8 individual samples. p
Figure : High Resolution (HR) Method Parameters with HTT configuration. p
Figure : High-Speed (HS) Method with HTT configuration for up to 0 individual samples p
Figure : High-Speed Method with HTT configuration for up to 8 individual samples p
Results and Discussion HR and HS methods using the HTT setup To demonstrate assay repeatability using the HTT setup, the SDS-MW Size Standard was used. For the HR and HS methods, 8 sample replicates were injected using the conventional (C)-tray array and 8 more samples for the HTT configuration. Figure 7 shows profiles obtained in the experiments with a) HR method and b) HS method. It is evident that both tray setups produce the same analyte profile and that no ghost peaks, artifacts, or any other spurious signal is present in the HTT configuration. This result demonstrates that the HTT setup can be implemented with high-sensitivity or high-resolution methods, therefore the IgGpurity/heterogeneity or the SDS-MW applications could be run under these experimental conditions. Application of the HR and HS Methods with the IgG/Purity Heterogeneity Assay Using the HTT configuration, separation reproducibility of the HR and HS methods was evaluated. Eight replicates of the IgG control standard were injected under non-reducing and reducing conditions. Under non-reducing conditions (Figure 8) the peaks observed correspond to the 0 kda internal standard, the intact antibody (IgG) and the non-glycosylated heavy chain (NG). In addition, impurities that are known to be present in the IgG control standard, such as heavy-heavy (HH) and 2 heavy light chain (2HL) are resolved from the whole antibody using either method. Under reducing conditions (Figure 9), the peaks observed correspond to the 0 kda internal standard (IS), the IgG light chain (LC), IgG non-glycosylated heavy chain (NG) and the IgG heavy chain (HC). As in the previous case, all peaks can be easily identified using either method. Figure 7: Repeatability of HR and HS Methods using Conventional-tray and HTT setups. Stacked data illustrating 8 separations per tray setup using the a) HR method or b) HS method. To facilitate easier analysis, electropherograms were offset by 0.02 AU. Samples were alternately injected using the conventional (C) or the HTT setup. The order in which the samples were injected is indicated by the number. Figure 8: Comparison of HR and HS methods with the HTT setup for IgG Control Standard under Non-reducing Conditions. Peak identification: 0 kda is the Internal Standard, IgG is the intact antibody, NG the non-glycosylated heavy chain, HH and 2HL are the heavy-heavy (HH) and 2 heavy light chain impurities. p 7
Table 2. Accuracy and Repeatability of the HS and HR Methods under Non-Reducing Conditions Parameter Migration Time Area Percent Method HR HS HR HS Injection NG IgG NG IgG NG IgG NG IgG 2.2 27.0.2. 7.9 88. 7.7 88.27 2 2. 27...8 7.88 88.2 7.7 88.28 2.70 27.9.8.2 7.87 88. 7.87 88. 2.77 27.2.2. 7.89 88. 7.7 88.2 2.8 27.2..8 7.88 88.8 7.7 88.2 2.89 27.8.8.7 7.89 88. 7.7 88.7 7 2.9 27.0.0.7 7.79 88. 7. 88. 8 2.0 27.07.29. 7.88 88.9 7.77 88.2 Mean 2.7 27.2.0.29 7.87 88. 7.7.29 SD 0.2 0. 0.08 0.08 0.0 0.0 0.0 0.08 %CV 0. 0. 0.2 0. 0. 0.0 0.80 0. Table. Accuracy and Repeatability of the HS and HR Methods under Reducing Conditions Figure 9: Comparison of HR and HS methods with the HTT setup for IgG Control Standard under Reducing Conditions. Peak identification: 0 kda is the Internal Standard, LC is the Light Chain, NG the Non-glycosylated heavy chain and HC the Heavy Chain. Migration times and area percent were determined for the main identified peaks (NG, intact IgG, LC, and HC) (Tables 2 and ). These results indicate that the evaluated parameters show high reproducibility for 8 separations using either the HR or the HS method. Reproducibility for migration time was always less 0. %CV and lower than.7 %CV for the peak area percent. As expected, resolution was slightly lower in the HS mode due to the somewhat shorter capillary length; however adjacent peak pairs with close migration times, such as NG/IgG or NG/HC, can be resolved and unequivocally identified and quantified. This demonstrates that the HS method can be used with complete confidence under standardized and validated conditions. Molecular Weight Estimation of Myoglobin (Myo) and Bovine Serum Albumin (BSA) The SDS-MW analysis kit is designed for the separation and estimation of molecular weight (MW) of a protein in a range between 0 and 22 kda. The use of the HS method, for this experiment, was compared to the conventional HR method. The HTT setup was used for all experiments. The molecular weight of an unknown protein can be estimated by making a comparison against a standard curve created using proteins of known sizes. For this reason, a protein sizing standard mixture was injected in parallel to the Myo and BSA samples. The sizing standard reagent contained six proteins: 0, 20,, 0, 00 and 22 kda. Sample mixtures containing the internal standard (IS), Myo and BSA were treated under reducing or non-reducing conditions and three replicates of each condition were analyzed using the PA800 Plus instrument (Figure 0). In all cases, analyte peaks were well resolved using either the high speed or the high resolution method. Molecular weights for Myo and BSA were estimated using the qualitative analysis feature in the 2 Karat p 8
Control and Analysis Software. Results listed in Table indicate high reproducibility (%CV < 0.2, n = ) for the MW estimation of Myo or BSA, under non-reducing and reducing conditions. Similarly, the difference of the MW values determined by either method (HS or HR) was minimal with an absolute value difference lower than 0.2 kda. Therefore, both methods can be used under optimized experimental conditions producing comparable results. Table. Determination of Molecular Weight for Myoglobin and Bovine Albumin Serum (in kda) Non-reduced Reduced HR method HS method HR method HS-Method Injection Myo BSA Myo BSA Myo BSA Myo BSA.9.9.0.720.989 8.7.9 8. 2.99.9.0.20.989 8.7.87 8.829.99.9.9.299.989 8.8.808 9.02 Mean.9.9.99..989 8.7.87 8.72 SD 0.020 0.000 0.00 0.2 0.000 0.09 0.09 0. %CV 0. 0.000 0.2 0.2 0.000 0.00 0.0 0. Conclusions The IgG Purity/Heterogeneity and SDS-MW assays can be performed using a high speed (HS) separation method with an inlet to detection window of 0.0 cm, using the capillary cartridge in the right to left configuration. In comparison to the more highly resolving conventional High Resolution (HR) method, results are achieved faster saving assay time. The use of a high throughput tray (HTT) setup in order to maximize the number of samples per run sequence does not cause deterioration in the separation. The combination of the HS method with the HTT setup is an alternative when in need of higher throughput sample analysis. References Figure 0: Determination of Protein Molecular Weight using HR and HS methods. Peak identification: 0 kda is the Internal Standard, Myo is myoglobin and BSA is Bovine Serum Albumin. Three injections under reduced and non-reduced conditions were analyzed. A size standard mixture was run before to inject the samples with the HS or HR-methods.. PA 800 Plus Pharmaceutical Analysis System-IgG Purity/Heterogeneity Assay Application Guide. SCIEX part number A97AD, January 20. 2. PA 800 Plus Pharmaceutical Analysis System-SDS-MW- Analysis Assay Application Guide. SCIEX part number A970AD, January 20 20 AB SCIEX. For Research Use Only. Not for use in diagnostic procedures. AB SCIEX is doing business as SCIEX. The trademarks mentioned herein are the property of AB SCIEX Pte. Ltd. or their respective owners. AB SCIEX is being used under license. Document number: RUO-MKT-02-2-A p 9