Abstract

Abstract

This technical note describes a process for rapid CE-SDS purity analysis of protein samples utilizing an automated pipetting workstation and high-throughput capillary electrophoresis. Sample preparation was completed using a Biomek i5 Span-8 robotic workstation (Figure 1) from Beckman Coulter^®.

CE-SDS is used to analyze drug purity, integrity and stability of therapeutic molecules, such as mAb. CE-SDS purity assays are required at different stages of the antibody development lifecycle, including stages of screening through characterization, production process development, quality control (QC) and product release.

The BioPhase 8800 system (Figure 1) is a high-throughput capillary electrophoresis platform that enables separation and detection on 8 capillaries simultaneously. This system can therefore significantly improve the speed of method development and sample analysis throughout the product lifecycle.

All sample preparation steps, such as heating and mixing, were fully automated, reducing manual operation time and minimizing potential errors caused by manual operation.

The sample separation and data analysis were performed using the SCIEX BioPhase 8800 system. The CE-SDS analysis of 96 mAb samples under reducing conditions was completed in 6.9 hours, compared to the 12 hours that would be required for analysis on a single capillary system. The inter- and intracapillary results varied by <0.23% for corrected peak area percentage (CPA%) and <0.36% for relative migration time (RMT) for heavy chain (tables 4 and 5). These results demonstrate the robustness and reliability of the workflow, from sample preparation to CE analysis.

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Key-features

Key features of the CE-SDS analysis of mAbs

• High-throughput sample preparation:  Nearly 2X-fold increase in sample throughput reduced total analysis time for 96 mAb samples to 6.9 hours from 12 hours
• Automated sample preparation: Hands-free sample preparation of 96 samples was performed in <1.5 hours
• Robust and reproducible analytical performance: Reach exceptional intra- and inter-capillary performance and reproducibility, with <0.25% %RSD for CPA% and <0.36% for migration time of heavy chain
• Streamlined workflow: Automated preparation of an integrated assay using a multi-channel CE data acquisition system

Introduction

Introduction

The ability to analyze 8 samples in parallel on the BioPhase 8800 system using a CE-SDS lightning method enables a 1.5- fold increase in throughput per channel. As a result, 96 samples can be processed in only 6.9 hours.

Manual sample preparation processes are labor-intensive and a potential source of human error and user variability. Three methods were developed using the Biomek i5 Span-8 automated workstation from Beckman Coulter (Figure 1). One method was developed to process up to 96 samples before CE-SDS analysis on the BioPhase 8800 system, whereas the other 2 methods were developed to prepare the reagent inlet and outlet plates using the CE-SDS kit.

The sample preparation for CE-SDS analysis under reducing conditions requires the samples to be heat denatured with SDS and β-mercaptoethanol (β-ME). Therefore, consistent reagent and sample transfers, reagent mixing and heating are important downstream for accurate protein quantitation and reliable separation. Automating the sample preparation process can potentially address these issues.

Methods

Methods

Materials:  The BioPhase CE-SDS Protein Analysis kit (P/N: C30085), including the SDS-MW separation gel buffer, 10 kDa internal standard, CE-grade water and basic and acidic washes, was from SCIEX (Framingham, MA). The β-ME (P/N: M3148- 25ML) was from Sigma Aldrich (St. Louis, MO).

The sample was a proprietary mAb at a 5 mg/mL concentration.

CE instrumentation:  The BioPhase 8800 system (P/N: 5083590) was from SCIEX (Framingham, MA).

Data acquisition and analysis software:  Data analysis was performed using the BioPhase 8800 software, version 1.0.

Instrumentation for automated sample preparation:  The Biomek i5 Span-8 automated workstation from Beckman Coulter Life Sciences (Indianapolis, IN) was controlled by Biomek i5 acquisition software, version 5.1 Sample preparation and addition of reagents were performed with the Biomek i5 Span-8 automated pipetting workstation equipped with a 96-well plate holder, custom sample plate heating module, 1070 µL and 90 µL automated pipette tips, 1.5 mL tube rack, 19 mL reagent tank, tip recovery bin (TR1) and tip cleaning station (W1). A diagram of the workstation deck is shown in Figure 2 with the position of each reagent marked. Figure 3 shows the workflow steps required to prepare the inlet and outlet sample and reagent plates for downstream analysis on the BioPhase 8800 system.

Automated sample preparation: The Biomek i5 Span-8 automated workstation was used to transfer 85 µL aliquots of sample buffer from Reagent Tank C (Figure 2) into the BioPhase 8800 sample plate wells. Next, 10 µL of the mAb sample, 2 µL of 10 kDa internal standard and 5 µL of β-ME were transferred to each well of the sample plate. This sample mixture was thoroughly homogenized using the tip mixing technique. Upon addition of the sample mixture, the method paused to allow the operator to manually set a heat-resistant film over the plate. After resuming the method, the sample plate was transferred to an on-deck custom heating plate. The sample was heat denatured and reduced at 70°C for 10 min, completing the automated sample preparation. The user then manually removed the plate from the deck and centrifuged it at 4 g for 4 min. The sample plate layout matched the sample plate design, as shown in Figure 4 (A1).

Figure 2. Diagram of the Biomek i5 Span-8 workstation deck.  A) BioPhase 8800 sample plate. B) A 1.5 mL tube rack for 10 kDa internal standard, β - ME and sample solution. C) Reagent tank with sample buffer in the first column. D) BioPhase 8800 sample outlet plate. E-H) Loading position for the 90 µL tips. I) Loading position for the 1070 µL tips. J) BioPhase 8800 buffer inlet plate. K) Reagent tank including basic wash, acidic wash, SDS separation gel and CE-grade water (in order from left to right). L) BioPhase 8800 buffer outlet plate.

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Reagent plate preparation: The buffer reagent inlet, outlet and sample outlet plates were prepared using the Biomek i5 Span-8 automated workstation using the layout shown in Figure 4 (A2, B1 and B2). Table 2 shows the reagent color codes, location and volumes used in this work. The method took approximately 22 minutes to prepare the outlet plates.

Figure 4. BioPhase 8800 sample tray, sample outlet tray, buffer inlet tray and buffer outlet tray reagent placement. A1) Inlet sample plate. A2) Outlet sample plate. B1) Inlet reagent plate. B2) Outlet reagent plate.

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The high-throughput CE-SDS method setup: The capillary conditioning method (Figure 5A) was performed at the beginning of the sequence. Figure 5B shows the details of the CE separation method. The shutdown method (Figure 5C) was executed at the completion of the sequence to prepare the capillary cartridge for storage. UV detection was performed at 220 nm. The separation cartridge and sample compartment temperatures were set to 25°C.

Results

Results and discussion

Reproducibility of the CE-SDS analysis of 96 reduced mAb samples

The sample and buffer plates prepared by the Biomek i5 Span-8 automated pipetting workstation were manually transferred to the BioPhase 8800 system for CE-SDS analysis. Figure 6 shows the overlay of the 96 electropherograms. The reproducibility of the CPA% and the RMT of the light chain (LC), heavy chain (HC) and non-glycosylated heavy chain (ng-HC) were calculated using the BioPhase 8800 software (Table 3). The %RSD values for RMT were 0.09%, 0.14% and 0.15% for LC, HC and ng-HC, respectively.

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The %RSD values for CPA% were 0.35%, 0.17% and 3.24% for LC, HC and ng-HC, respectively. These results demonstrate that combining the Biomek i5 Span-8 automated workstation and the BioPhase 8800 system provides robust and reproducible analytical results.

Inter- and intra-capillary precision:  Representative analyses were performed using the heavy chain peak as an example. Table 4 shows the average inter- and intra-capillary %CPA values calculated for the HC peak following the analysis of 96 samples. The rightmost column of Table 4 shows the intracapillary reproducibility across runs, whereas the bottom row of the table shows the inter-capillary reproducibility. The overall reproducibility for both intra- and inter-capillary was ≤0.22%. Similarly, Table 5 shows the average inter- and intra-capillary values calculated for RMT of the HC peak following the analysis of 96 samples. The rightmost column of Table 5 shows the intracapillary reproducibility and the bottom row of the table shows the inter-capillary reproducibility of RMT. The overall reproducibility for both intra- and inter-capillary was <0.36% for RMT. Together, these results demonstrate the exceptional precision of the automated sample preparation, compared to manual sample preparation.¹

Figure 5. Method setup parameters for the BioPhase 8800 system for analyzing protein purity. A) Pre-conditioning method. B) Separation method. C) Shutdown method.

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Figure 6. CE-SDS electrophoresis of 96 reduced mAbs. The A-H designation indicates unique capillaries and the numbers 1-12 denote the well positions.

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Conclusion

Conclusion

• This streamlined workflow combined with multi-capillary electrophoresis offers:

  • Ideal CE-SDS solution for mAb purity analysis, especially if large sample sets must be analyzed
  • A full 96-well plate preparation completed in 1.5 hours and CE separation completed in <7 hours
  • Significantly reduced manual lab work
  • Increased consistency of sample preparation and eliminated variability due to human errors

• High degree of reproducibility for both intra- and inter-capillary separations: The %RSD values for RMT were 0.09%, 0.14% and 0.15% for LC, HC and ng-HC, respectively. The %RSD values for CPA% were 0.35%, 0.17% and 3.24% for LC, HC and ng-HC, respectively.

• An all-inclusive BioPhase CE-SDS Protein Analysis kit: Contains the reagents required to process the sample and prepare the reagent inlet and outlet plates required for CE analysis

References

References

1. Intermidate precision study of capillary electrophoresissodium docedyl sulfate (CE-SDS) assay on the BioPhase 8800 system. SCIEX technical note, RUOMKT-02-14780-A.