High throughput CE-SDS protein analysis using fully automated sample and reagent preparation workflow
Abstract
This application note describes a fully automated CE-SDS (capillary electrophoresis sodium dodecylsulfate) workflow for preparing therapeutic biologic samples on the Biomek i5 MC liquid handler (Figure 1A) using the BioPhase CE-SDS Protein Analysis kit (Figure 1B), followed by separation and analysis on the BioPhase 8800 system (Figure 1C).
The CE-SDS method separates proteins by size to assess purity, heterogeneity, and impurities while streamlining sample and reagent plate preparation for high-throughput environments.
The workflow prepares 8 to 96 samples in increments of eight through a single user interface, increasing throughput, improving consistency, reducing hands-on time, and minimizing pipetting errors.
For broader automation, while not used in this work, the optional BioPhase Instrument API* enables external software to monitor instrument status, track sequence progress, and execute supported instrument and sequence operations.
Key features
- Automated CE-SDS workflow: Automates reduced mAb sample and reagent plate preparation on the Biomek i5 MC Workstation, followed by analysis on the multi-capillary BioPhase 8800 system.
- High-throughput sample processing: Processes 8 to 96 samples in increments of eight, supporting efficient characterization of large sample sets.
- Reduced hands-on time and error risk: Reduces manual steps and pipetting errors with guided setup and deck verification.
- Excellent reproducibility: Delivers consistent CE-SDS performance across 96 samples, with <0.24% RSD for RMT and <0.38% RSD for heavy chain CPA%.
- Improved efficiency: Prepares up to 96 samples with 15 minutes of human hands-on time (for deck preparation, using a 300 µL MC head), two user interactions, and ~3 hours total runtime.
- Optional BioPhase Instrument API*: Enables software-based monitoring and run control for integration into automated laboratory workflows
Introduction
Therapeutic biologics have revolutionized modern medicine and now represent a multi-billion-dollar business, with hundreds currently approved by the FDA and other international regulatory agencies and thousands more in the development pipeline. These molecules range from traditional monoclonal antibodies, antibody-drug conjugates, multi-specific antibodies, fusion proteins, nanobodies and peptibodies to cell and gene therapy products such as lipid nanoparticles and adeno-associated viruses. In the fast-paced environment of upstream bioprocessing of therapeutic biologics, many samples must be prepared and analyzed to determine and quantify the purity and heterogeneity by chromatographic or electrophoretic means. Such a high number of samples tends to quickly become a bottleneck.
Therefore, to address the throughput limitation in the analytical characterization of intermediate samples, this application note demonstrates the fully automated preparation of reduced monoclonal antibodies using the BioPhase CE-SDS Protein Analysis kit and the Biomek i5 MC liquid handler, with downstream CE separation using the BioPhase 8800 system. This method also allows for preparing all reagent plates used for the electrophoretic separation by CE-SDS.
The BioPhase CE-SDS Protein Analysis kit is used to separate reduced and non-reduced protein samples by size and quantify the heterogeneity and impurities that might exist.
While this focuses on the CE-SDS separation and sample prep of a monoclonal antibody under reduced conditions, this workflow can be applied to other biologic therapeutic proteins under reduced and non reducing conditions. Once denatured and reduced, the sample is introduced into the capillary by voltage injection and separated using a bare fused silica capillary containing a replaceable SDS polymer matrix, which provides the sieving selectivity for the separation.
The automated method for the BioPhase CE-SDS Protein Analysis kit on the Biomek i5 MC liquid handler can prep from 8 to 96 samples in increments of 8. The user can select the preparation of samples and sample inlet tray, reagent inlet and outlet trays and the sample outlet tray. The sample outlet, reagent outlet and inlet tray prep sections can be operated from start to finish with a single user interface to keep labware and reagents on deck. For the sample prep portion, the liquid handler is paused so the user can add a sealing film on the processing plate before the robotic arm transfers the plate to the Peltier for heating. In this application note, we will demonstrate the performance of the BioPhase CE-SDS Protein Analysis kit on the Biomek i5 MC Workstation automated workflow.
Materials
Consumables: The BioPhase CE-SDS Protein Analysis kit (PN C30085), including the SDS-MW separation gel buffer, 10 KDa internal standard, basic wash, acidic wash, sample buffer, IgG control standard (PN 391734) and SDS-MW size standard (PN A22196) were from SCIEX (Marlborough, MA). The BioPhase BFS capillary cartridge - 8 x 30 cm (PN 5080121) and the BioPhase Plate Pack Starter kit (PN 5080311) were from SCIEX (Marlborough, MA). Multi-Therm shaker incubator (PN H5000-H) was from Benchmark Scientific (Sayreville, NJ) . The β-ME (PN M3148-25ML) was from Sigma Aldrich (St. Louis, MO). USP monoclonal IgG system suitability (PN 1445550) from USP (Rockville, MD) was used as reference standard for testing. PureAmp Pre-Cut Sealing Film - Aluminum Membrane, (PN P1001-A) from Universal Medical (Oldsmar, FL)
Instrumentation:
The Biomek i5 MC workstation features include:
- 96 head (1 µL - 1200 µL pipetting capability). The Multichannel pipetting head and flexible configurations to increase walk-away time
- 360˚ rotating gripper with offset fingers
- Guided Labware Setup (GLS)
- DeckOptix Final Check to ensure accurate system setup
- Reagent volume calculations
- 96 channel tips washing for sample processing and bead-based cleanups
- High deck capacity with 25 positions
The BioPhase 8800 system (PN 5083590F) features include:
- 8-capillary cartridge for simultaneous CE analysis
- Equipped with UV and laser-induced fluorescence (LIF) detection modes; both detectors can be used in the same sequence
- Easy and intuitive front panel control software
- User-friendly method and sequence editor and data analysis software
Sample and plate preparation: To demonstrate method capabilities for a 96-sample preparation on a Biomek i5 MC workstation (Figure 2), we started with 95 μL of USP IgG SS standard in a 96-well plate. To the sample 2 μL of internal standard and 5 μL of β-ME were added individually. The plate was then sealed with a film and placed back on deck. It was then heated to 70°C for 10 minutes. 90 μL of these samples were then transferred to the sample inlet tray. The sample inlet tray was centrifuged at 177×g for 20 minutes and then placed into the BioPhase 8800 system for electrophoretic separation. For the reagent inlet plate, all reagents were pipetted column-wise as provided in the user guide. For reagent outlet, a single tip is used for each reagent. For sample outlet tray, a tip is used for each well that needs to be filled (depending on the sample number). The reagent inlet and outlet trays and sample outlet trays were prepped with the Biomek i5 MC workstation and then placed directly into the BioPhase 8800 system. The capillary conditioning was performed as per the user guide followed by the separation. To determine intra- and inter-capillary reproducibility, we analyzed the corrected peak area (CPA) %, relative migration time (RMT) of the species of the reduced control IgG, the ratio of CPA between the heavy chain (HC) and light chain (LC) and the resolution between the peaks of the non-glycosylated heavy chain (ng-HC) and HC
Discussion
Automated method
Automation provides increased efficiency and reduction in human errors, with minimal hands-on time (Table 1). Please note, these timings are for a 300 µL MC head. With a switch to 1200 µL MC head, the preparation time can be reduced.
Intra- and inter-capillary reproducibility using IgG control standard
We demonstrate an inter-capillary reproducibility better than 0.24 relative standard deviation (RSD) for RMT and 0.38 for CPA% for the HC peak of reduced IgG control standard. For intra-capillary reproducibility, our results showed an RSD for RMT below 0.28% and between 0.21% and 0.47% for CPA%.
Figure 5A shows the overlayed electropherograms of the IgG control standard for all 96 samples, and Figure 5B illustrates a typical profile of the reduced USP monoclonal IgG system suitability from USP.
Corrected peak area (CPA) %: The CPA% was determined automatically by the BioPhase software. Briefly, the CPA% was calculated by dividing the peak area by the velocity of the corresponding peak. The average values for the CPA% of the major species found in the reduced IgG control standards (LC, ng-HC and HC) are displayed in Table 2. Consistent values for CPA% were observed for the reduced form of the IgG control standards across all 96 samples.
HC/LC ratio: The average intra-capillary values are shown in Table 3 and indicate the consistency of the data. A %RSD of the HC/LC ratio between 0.24 and 0.44% was observed for intra-capillary and 0.37% for inter-capillary, indicating good reproducibility (Table 2).
Migration Time (MT) and RMT: Table 3 summarizes the intra- and inter-capillary average values for MT. Overall, the average values (N=96) for MT were consistent for inter- and intra-capillaries, indicating the assay's specificity and reproducibility, and the cartridge cooling features of the BioPhase 8800 system. Table 3 highlights a %RSD better than 0.8% for the MT for the 3 major species of reduced IgG control standard. The RMT for the 3 species and the 10 KDa marker are plotted for all 96 samples in Figure 6A.
Resolution ng-HC and HC: Resolution between the ng-HC and HC is a critical parameter because its presence is considered a product-related impurity, as it alters the stability and effector functions of the final therapeutic product. Therefore, it is essential to separate and quantify this variant consistently. The mobility of these species is very similar, and therefore it is necessary to monitor resolution and specificity during the CE-SDS separation under reduced conditions.
* available separately and under the API EULA T&Cs
Conclusions
- Automated sample and reagent inlet and outlet plate preparation on the Biomek i5 MC workstation eliminates labor-intensive bench work and provides a higher throughput for SDS analysis with consistent results when combined with the BioPhase 8800 system.
- Automated sample preparation yielded excellent intra-capillary reproducibility data for migration time, relative migration time and corrected peak area percentage.
- Inter-capillary reproducibility values for % RSD for CPA% were less than 0.47, 2.24 and 0.38 for LC, ng-HC and HC respectively.
- Inter-capillary reproducibility values for % RSD for RMT were less than 0.27, 0 and 0.21 for LC, ng-HC and HC respectively.
- The BioPhase 8800 was operated using standard BioPhase software for instrument control and data acquisition. For greater automation, even though the API* was not evaluated in this work, SCIEX offers the BioPhase Instrument API*, which allows external Figure 6: (A) Relative Migration Time (RMT) for the 10 KDa marker, LC, ng-HC and HC for all 96 samples. (B) Resolution between the ng-HC and HC peak for 96 samples
- software to monitor instrument status, trigger and track a sequence progress, and integrate the system into broader lab automation workflows.