abstract

Abstract

As adeno-associated virus (AAV) gene therapies progress toward clinical and commercial viability, robust analytical workflows are essential for ensuring product quality and regulatory compliance.^1,2 Capillary electrophoresis (CE) platforms, such as the BioPhase 8800 system from SCIEX, offer integrated solutions for multi-stage critical quality attribute (CQA) analysis across the AAV lifecycle-from plasmid critical starting material to AAV product.3 This technical note evaluates the performance of a newly integrated native fluorescence (NF) detection on the BioPhase 8800 system for AAV capsid protein analysis using a CE-SDS workflow. Compared to traditional ultraviolet (UV) detection, native fluorescence (NF) detection provides improved baseline stability and enables label-free detection of viral proteins (VPs), thus simplifying sample preparation and enhancing data processing. While its sensitivity does not exceed that of laser-induced fluorescence (LIF) detection, NF detection offers a balanced alternative with superior repeatability and linearity. Three AAV samples, including serotype 8, serotype 9, and a self-complementary (sc) variant, were analyzed to compare detection modes. Results demonstrate that NF detection enhances workflow efficiency and analytical robustness, supporting its utility in high-throughput AAV protein characterization.

Figure 1: Capsid protein analysis of AAV8-CMV-GFP using CE-SDS on the BioPhase 8800 system with three detection modes. Linearity plots are shown for NF (left), UV (middle), and LIF (right). All three detection modes demonstrate excellent linearity across the tested titer range, with correlation coefficients (R²) > 0.99. NF detection shows a threefold improvement in limit of detection (LOD) compared to UV, while LIF achieves the highest sensitivity with an LOD of ~1×109 GC/mL. NF provides a label-free alternative with improved baseline stability and precision, while LIF remains optimal for ultra-low titer samples.

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Keybenefits

Key benefits of CE-SDS with NF detection for AAV capsid protein analysis

• Extension of AAV QC workflow capabilities: NF extends the existing characterization capabilities for plasmid to AAV capsid protein and genome titer with an additional detection mode, offering flexibility for protein analyses in AAV drug development and QC.
• Superior performance compared to UV detection: NF offers ~3× higher sensitivity, tighter %RSDs for capsid protein purity (CPA%), and faster data processing due to a stable, flat baseline, especially beneficial for detecting low-abundance VP peaks and impurities.
• Label-free workflow vs. LIF detection: NF eliminates the need for fluorescent dye labeling and thus reduces variability from dye labeling.

Introduction

Introduction

As gene therapy continues to advance, the need for scalable, reproducible, and regulatory-compliant analytical workflows for AAV drug production has become increasingly critical.^1,2 CE platforms now offer integrated solutions for comprehensive CQA analysis across the AAV lifecycle, from plasmid DNA integrity to capsid protein characterization, within a single system.³

The SCIEX BioPhase 8800 system supports a suite of CE-based assays that streamline quality assessments at multiple stages of AAV development, which can reduce technical transfer challenges, accelerate scale-up, and ensure consistency in AAV drug quality. As illustrated in Figure 2,  the system enables high-resolution purity analysis of plasmid DNA using the DNA 20 kb Plasmid and Linear kit,⁴ RNA integrity evaluation via the RNA 9000 Purity & Integrity kit,⁵ and capsid protein purity analysis and VP ratio determination through the CE-SDS Protein Analysis kit.⁶ Additionally, a novel approach allows for quantitation of full, partial, and empty capsids by integrating genome titer and capsid titer data.⁷

Figure 2. Streamlined CE-based analytical workflows for AAV drug production using the BioPhase 8800 system.Representative electropherograms illustrate the multi-stage quality control enabled by a single platform: (A) Separation of plasmid isoforms using the DNA 20 kb Plasmid and Linear kit; (B) RNA integrity assessment with the RNA 9000 Purity & Integrity kit; (C) and (D) Capsid protein analysis with the CE-SDS Protein Analysis kit using UV and LIF detection, respectively. Additionally, quantitation of full, partial, and empty capsids is derived from genome and capsid titer measurements. Together, these assays demonstrate a unified platform approach for comprehensive AAV CQA analysis.

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Methods

Traditionally, CE-SDS with UV detection has been widely used for AAV capsid protein analysis. While effective for high-titer samples, UV-based workflows often suffer from baseline fluctuations due to non-analyte absorbance and gel buffer heterogeneity, complicating peak integration and slowing data analysis. Moreover, UV detection lacks the sensitivity required for low-titer or in-process samples. CE-SDS with LIF detection offers improved sensitivity but introduces complexity through dye labeling, which can affect assay accuracy due to variability in dye binding.

This technical note evaluates the performance of a newly integrated NFD on the BioPhase 8800 system. The NFD leverages the intrinsic fluorescence of aromatic amino acids, primarily tryptophan, to enable label-free detection of VPs with enhanced sensitivity and stable baselines compared to UV detection. This eliminates the need for dye labeling and simplifies sample preparation relative to LIF detection.

In this study, three AAV samples (serotype 8, serotype 9, and a self-complementary construct) were analyzed using CE-SDS coupled with UV, LIF, and NF detection on the BioPhase 8800 system with NF. Comparative results demonstrate that NFD provides improved sensitivity, more stable baselines, and simplified data processing relative to UV detection, while avoiding the labeling requirements of LIF. The integration of NFD expands the BioPhase 8800 system’s analytical capabilities, offering a more efficient and precise workflow for AAV capsid protein analysis.

Methods

Materials: The BioPhase CE-SDS Protein Analysis kit (P/N: C30085), the BioPhase BFS capillary cartridge - 8 x 30 cm (P/N: 5080121), and the BioPhase sample and reagent plates (4,4,8) (P/N: 5080311) were from SCIEX (Marlborough, MA). (P/N: 5080311) were from SCIEX (Marlborough, MA). The β-mercaptoethanol (β-ME) (P/N: M3148-25ML) was from MilliporeSigma (St. Louis, MO). The Chromeo P503 dye (P/N: 15106) was from ACTIVE MOTIF (Carlsbad, CA). Dithiothreitol (DTT, P/N: V3151) was from Promega (Fitchburg, WI). 10x Phosphate Buffered Saline (PBS) (P/N: AM9624) was from Thermo Fisher Scientific (Waltham, MA).

Samples: The AAV8-CMV-GFP (P/N: SL100833), AAV9-CMV-LacZ (P/N: SL100868) and scAAV9-GFP (P/N: SL100840-sc) were from SignaGen Laboratories (Rockville, MD). And these three samples were used for the capsid purity analysis and viral protein ratio comparison of 3 different detection ( UV, LIF, and NF) modes. The packaged AAV8 of pAV-CMV-GFP 1.50x10¹³ GC/mL) was from Charles River (Rockville, MD) and was used for comparison of the linearity, LOD, and LOQ determination of the 3 different detection modes (UV, LIF, and NF).

Sample preparation for AAV capsid protein analysis by CE-SDS-UV and CE-SDS-NF on the BioPhase 8800 system with NF: 10 μL of AAV sample solution was mixed with 10 μL of sample buffer from the BioPhase CE-SDS Protein Analysis kit and 3 μL of β-ME and incubated at 70°C for 10 minutes. After the samples were cooled to room temperature, 77 μL of DI water was added to the mixture. The diluted mixture was transferred into the appropriate well of the sample inlet plate. Analysis was then performed on the BioPhase 8800 system with NF using separation conditions described in the application note of the BioPhase CE-SDS Protein Analysis kit.⁸

Sample preparation for AAV capsid protein analysis by CE-SDS-LIF on the BioPhase 8800 system with NF: The AAV sample was mixed with 10 μL of sample buffer from the BioPhase CE-SDS Protein Analysis kit and 2 μL of 1M DTT and incubated at 70°C for 10 minutes. After samples were cooled to room temperature, 1 μL of 1 mg/mL Chromeo P503 dye (dissolved in methanol) was added, and the mixture was incubated at 70°C for another 10 minutes. After the samples were again cooled to room temperature, 77 μL of water was added to the mixture. The diluted mixture was transferred into the appropriate well of the sample inlet plate. Analysis was then performed on the BioPhase 8800 system with NF using separation conditions described previously.⁶

Sample preparation for AAV linearity analysis by CE-SDS-UV and CE-SDS-NF on the BioPhase 8800 system with NF: The packaged AAV8 of pAV-CMV-GFP (1.50 x 10¹³ GC/mL) was serially diluted 2-fold with 1x PBS. A total of 14 dilutions were made with AAV concentration in a range of 1.50 x 10¹³ GC/mL to 1.83 x 10⁹ GC/mL. Each diluted sample was prepared and analyzed following the sample preparation for AAV capsid protein analysis by CE-SDS-UV and CE-SDS-NF on the BioPhase 8800 system with NF.

Sample preparation for AAV linearity analysis by CE-SDS-LIF on the BioPhase 8800 system with NF: The packaged AAV8 of pAV-CMV-GFP (1.50 x 10¹³ GC/mL) was serially diluted 2-fold with 1x PBS. A total of 15 dilutions were made with AAV concentration in a range of 1.50 x 10¹³ GC/mL to 9.16 x 10⁸ GC/mL. Each diluted sample was prepared and analyzed following the sample preparation for AAV capsid protein analysis by CE-SDS-LIF on the BioPhase 8800 system with NF.

Instrumentation: The BioPhase 8800 system with UV/LIF/NF (P/N: 5314860) was from SCIEX. The Multi-Therm shaker incubator (P/N: H5000-H) was from Benchmark Scientific (Sayreville, NJ).

Software: BioPhase software package version 1.5 was used for creating run methods and sequences, and for data acquisition and processing.

Results

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Results and discussion

Capsid protein purity analysis of AAV8 using CE-SDS with three detection modes: Accurate characterization of AAV capsid proteins is essential for evaluating product quality, consistency, and potency in gene therapy applications.¹ The CE-SDS assay on the BioPhase 8800 system enables high-resolution separation of VPs, including VP1, VP2, VP3, and VP3’, and supports multiple detection modes to accommodate varying analytical needs. In this section, we compare the performance of three detection technologies-NF, UV, and LIF-in analyzing the capsid proteins of three representative AAV samples. Each method offers distinct advantages and limitations in terms of sensitivity, baseline stability, and quantitation accuracy. The following results highlight how these detection modes influence data quality and repeatability in CE-SDS-based capsid protein analysis.

Figure 3 presents the comparative analysis of AAV8-CMV-GFP capsid proteins using CE-SDS on the BioPhase 8800 system with three different detection modes: NF, UV, and LIF.

Panel A displays electropherograms (E-grams) of a single injection analyzed by each detection mode. All three methods successfully resolved the capsid protein components-VP1, VP2, VP3, and VP3′-with comparable peak profiles. However, distinct differences in baseline stability and sensitivity were observed. The UV detection trace shows noticeable baseline fluctuations, particularly in the 15–30 minute range, where the VP peaks and low-abundance impurity peaks migrate. These fluctuations complicate peak integration and obscure minor impurity peaks, especially those migrating between 15 and 20 minutes. In contrast, both NF and LIF detection modes exhibit flat, stable baselines, enabling more straightforward and automated peak integration. While NF improves sensitivity over UV, it does not match the sensitivity of LIF, which clearly detects low-abundance species with signal-to-noise ratios (S/N) greater than 10.

Panel B illustrates repeat injections of the same AAV8-CMV-GFP sample by each detection mode: NF (n=7), UV (n=7), and LIF (n=8). All three detection modes demonstrate excellent repeatability in migration time and peak shape for VP proteins. However, baseline consistency again differentiates the methods. UV traces show variability in baseline drift, while NF and LIF maintain consistent baselines across replicates, supporting more reliable integration and quantitation.

Table 1 summarizes the repeatability metrics for capsid protein purity analysis across detection modes. All methods yielded low relative standard deviations (RSDs) for VP migration times, confirming robust separation performance. NF detection produced comparable capsid protein purity percentages (corrected peak area percentage, CPA%) to UV, but with tighter RSDs, especially for low-abundance peaks, due to improved baseline stability. LIF detection provided the most consistent CPA% values, but its reliance on dye labelling introduces potential bias. Specifically, VP1 and VP2 peaks showed disproportionately high CPA% under LIF due to increased lysine content, which enhances dye binding with P503. This non-stoichiometric labeling can distort intensity-based quantitation, making normalization essential for accurate VP ratio determination.

To further validate these findings, the same CE-SDS experiment was conducted using two additional AAV samples: AAV9-CMV-lacZ and scAAV9-CMV-GFP. Although the electropherograms are not shown in this technical note, the repeatability results are summarized in Tables 2 and 3 below. Consistent with the AAV8-CMV-GFP data presented in Figure 3 and Table 1, all three detection modes demonstrated excellent repeatability in migration time and VP peak profiles. NF detection again showed improved baseline stability and tighter RSDs compared to UV, while LIF maintained the highest sensitivity but required dye labelling. These results reinforce the conclusion that NF detection offers a robust, label-free alternative for capsid protein analysis with performance comparable to LIF and superior to UV in terms of baseline stability and integration precision.

In summary, NF detection offers a label-free alternative with improved baseline stability and quantitation precision over UV, and comparable repeatability to LIF. While LIF remains the most sensitive method, its dependency on dye labelling introduces variability that must be accounted for in quantitative analysis. Future work will focus on normalization strategies to correct for dye-binding bias and improve accuracy in VP ratio determination across detection platforms.

Figure 3.   Capsid protein analysis of AAV8-CMV-GFP using CE-SDS on the BioPhase 8800 system with three detection modes. Panel A (left): Electropherograms of a single injection analyzed by NF detection (top), UV detection (middle), and LIF detection (bottom). All three methods resolve VP1, VP2, VP3, and VP3′ peaks with comparable profiles. NF and LIF show flat baselines, enabling easier and more accurate peak integration, while UV detection exhibits baseline fluctuations and lower sensitivity, missing low-abundance impurities. Panel B (right): Repeat injections of the same sample analyzed by NF (n=7, top), UV (n=7, middle), and LIF (n=8, bottom). All detection modes demonstrate excellent repeatability in migration time and peak shape. NF and LIF maintain consistent baselines across replicates, supporting robust quantitation and automated data processing.

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Table 1. Repeatability comparison of the AAV8-CMV-GFP capsid protein purity analysis using CE-SDS with UV, LIF, and NF detection on the BioPhase 8800 system. This table summarizes the relative standard deviations (RSD%) of migration times and capsid protein purity percentages (CPA%) for VP1, VP2, and VP3 across three detection modes.

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Table 2. Repeatability comparison of the AAV9-CMV-LacZ capsid protein purity analysis using CE-SDS with UV, LIF, and NF detection on the BioPhase 8800 system with NF. This table summarizes the relative standard deviations (RSD%) of migration times and capsid protein purity percentages (CPA%) for VP1, VP2, and VP3 across three detection modes.

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Table 3. Repeatability comparison of the scAAV9-CMV-GFP capsid protein purity analysis using CE-SDS with UV, LIF, and NF detection on the BioPhase 8800 system. This table summarizes the relative standard deviations (RSD%) of migration times and capsid protein purity percentages (CPA%) for VP1, VP2, and VP3 across three detection modes.

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Linearity assessment of VP3 quantitation across detection modes: Quantitative accuracy and sensitivity are critical for capsid protein analysis, especially when working with AAV samples across a wide range of titers. To evaluate the linearity of VP3 quantitation, a series of AAV8-CMV-GFP samples with varying genome copy (GC) concentrations were analyzed using CE-SDS on the BioPhase 8800 system with three detection modes: NF, UV, and LIF.

Figure 4 presents the linearity plots of VP3 capsid protein purity percentage (CPA%) versus AAV titer for each detection mode. The left panel shows the NF detection plot, the middle panel displays UV detection, and the right panel illustrates LIF detection. All three methods demonstrated excellent linearity, with R²> 0.99 across the tested concentration ranges.

Table 4 summarizes the linearity metrics, including the tested titer range, limit of detection (LOD), and limit of quantitation (LOQ), determined using S/N P-P calculated by the BioPhase 8800 software. For this specific AAV8-CMV-GFP sample, NF detection achieved a threefold improvement in LOD compared to UV, detecting down to approximately 1×10¹¹ GC/mL. LIF detection exhibited the highest sensitivity, with an LOD of 1×10⁹ GC/mL, making it suitable for ultra-low titer samples.

While LIF offers superior sensitivity, it requires fluorescent dye labeling, which introduces complexity and potential variability in quantitation due to non-stoichiometric dye binding. In contrast, NF detection provides a label-free, streamlined workflow with improved baseline stability and precision over UV, making it a practical alternative for routine capsid protein purity analysis. NF detection also enhances impurity detection and supports consistent peak integration, especially in low-abundance regions.

In summary, all three detection modes are suitable for quantitative analysis of VP3 across a broad titer range. NF detection stands out as a balanced solution, combining improved sensitivity over UV with the simplicity of label-free operation. LIF remains the method of choice for ultra-low titer applications where maximum sensitivity is required.

Casestudy

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VP ratio determination of AAV capsid proteins using CE-SDS with multiple detection modes: The ratio of viral proteins (VP1VP3) in AAV capsids is a critical quality attribute that reflects capsid integrity and assembly efficiency. While the theoretical ratio is approximately 1:1:10, actual measurements can vary depending on the AAV batch, assay type and the detection method used for a given assay type, i. e., CE-SDS analysis. In this study, three AAV samples-AAV8-CMV-GFP, AAV9-CMV-lacZ, and scAAV9-CMV-GFP-were analyzed using CE-SDS on the BioPhase 8800 system with three detection modes.

Table 5 summarizes the VP ratio results obtained from each detection mode. While all methods resolved VP1, VP2, and VP3 peaks, the calculated ratios differed significantly, particularly with LIF detection. This discrepancy is attributed to the inherent differences in detection principles:

UV detection (220 nm) measures absorbance from the peptide backbone, reflecting total amino acid content. It is simple and widely available but has lower sensitivity, making it less preferable for low-concentration samples. VP2 and VP3 may be underrepresented due to their lower amino acid counts.

LIF detection uses dyes such as Chromeo P503 that bind to lysine residues. While highly sensitive and suitable for low-titer samples, dye binding varies across VP proteins due to differences in lysine content. This can lead to overestimation of VP1 and VP2 relative to VP3.

NF detection relies on intrinsic fluorescence from tryptophan residues. It is a label-free detection technique and offers improved baseline stability. Since its sensitivity depends on tryptophan content, sensitivity will vary among VP proteins. VP3 and VP2 may be underrepresented if they contain fewer tryptophan residues.

To address these biases, normalization was applied using the known amino acid composition of VP1, VP2, and VP3 from the AAV8-CMV-GFP sequence. Specifically, the number of tryptophan (# of W), total amino acids (# of AA) and lysine (# of K) was used to adjust the intensity-based VP ratios (shown in Table 6).

After normalization, the VP ratios obtained from LIF detection were comparable to those from UV and NF detection. However, residual differences remained, likely due to factors such as protein folding, accessibility of dye-binding sites, and structural heterogeneity, which affect the dye binding efficiency of the VPs and fluorescence intensity.

These findings highlight the importance of understanding detection-specific biases when interpreting VP ratios. While NF detection offers a practical, label-free alternative with improved precision and baseline stability, LIF remains valuable for ultra-low titer samples, provided that normalization strategies are applied to correct for dye-binding variability.

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Conclusion

Conclusion

The integration of NFD into the CE-SDS workflow on the BioPhase 8800 system significantly enhances the analytical capabilities for AAV capsid protein characterization. By leveraging intrinsic fluorescence from tryptophan residues, NFD enables a label-free, high-precision, and time-efficient approach to VP analysis. This advancement complements the streamlined AAV drug production quality control process-from plasmid to capsid-by offering improved sensitivity, reproducibility, and operational simplicity. Below are the key features that highlight the value of NFD in this workflow:

• Integrated into streamlined AAV QC workflow: Extends CE-based quality control from plasmid DNA to capsid protein analysis, supporting end-to-end characterization on a single platform.
• Enhanced sensitivity over UV detection: Approximately 3× greater sensitivity for AAV samples compared to UV, enabling improved detection of low-abundance impurities and more accurate quantitation at lower titers.
• Improved precision for CPA% measurements: Delivers tighter %CVs for capsid protein purity due to consistent baseline and reliable peak integration, especially critical for low-abundance VP peaks.
• Time-saving during data processing: Stable, flat baseline simplifies peak integration, reducing manual effort and accelerating analysis-ideal for high-throughput workflows and DoE studies.
• Label-free workflow: Eliminates the need for fluorescent dye labeling required in LIF detection, minimizing variability from reagent inconsistencies, manual handling, and labeling efficiency differences between VPs.

References

References

1. Zwi-Dantsis L, Mohamed S, Massaro G, Moeendarbary E. Adeno-Associated Virus Vectors: Principles, Practices, and Prospects in Gene Therapy. Viruses. 2025 Feb 9;17(2):239. doi: 10.3390/v17020239. PMID: 40006994; PMCID: PMC11861813. Adeno-Associated Virus Vectors: Principles, Practices, and Prospects in Gene Therapy

2. Wang, JH., Gessler, D.J., Zhan, W.  et al.  Adeno-associated virus as a delivery vector for gene therapy of human diseases.  Sig Transduct Target Ther  9, 78 (2024). https://doi.org/10.1038/s41392-024-01780-w

3. Comprehensive critical quality attribute (CQA) analysis for AAV drug production by multiple workflows on a single CE platform. SCIEX technical note, MKT-34670-A

4. A new method for monitoring plasmid purity: Seamless method transfer and consistent results across single and multi-capillary systems. SCIEX technical note, MKT-32653-A

5. Genome integrity analysis of adeno-associated viruses (AAV) using multi-capillary gel electrophoresis. SCIEX technical note, RUO-MKT-02-13572-A

6. Acceleration of method optimization for AAV capsid purity analysis using multi-capillary electrophoresis platform. SCIEX technical note, RUO-MKT-02-13368-B

7. A  new approach for high-resolution full and empty AAV capsid analysis utilizing a high-throughput method for comprehensive AAV evaluation on a single CE platform. SCIEX technical note, RUO-MKT-02-15218-A

8. CE-SDS Protein Analysis Kit. SCIEX application guide, RUO-IDV-05-8662-D