Executive Summary

• Automated 384-Well Validation: Multus integrated PROGEN's Pan AAV Capsid Titer Assay with the Analytik Jena CyBio FeliX liquid handler, upscaling from 96 to 384-well format with maintained dynamic range, complete luminescence crosstalk elimination using a plate reader with aperture spoon, and a 11.4% mean inter-assay CV.
• Universal Serotype Coverage: The Lumit®-based assay demonstrated consistent, linear performance across eight common and novel AAV serotypes, including AAV-PHP.eb and AAV2.7m8, in a rapid no-wash, add-and-read workflow.
• Independent External Validation: Assay reproducibility, recovery, and inter-assay precision were confirmed independently by external testing in six additional laboratories.

Challenge

Screening media compositions for AAV manufacturing requires high-throughput capsid titer assessment across hundreds of formulations. Conventional ELISA-based titer methods are labor-intensive and ill-suited to automation, quickly becoming the bottleneck in large-scale screening programs. The rapid expansion of engineered AAV variants further demands a sngle universal assay with pan-serotype coverage that can integrate into a 384-well automated workflow without sacrificing accuracy.

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Project Overview

Multus partnered with PROGEN to validate the Pan AAV Capsid Titer Assay for use in an automated high-throughput format, targeting:

1. Confirm dynamic range is maintained when scaling from 96 to 384-well format.
2. Assess and resolve luminescence crosstalk between adjacent wells in opaque 384-well plates.
3. Evaluate matrix effects of clarified HEK293 cell lysates across a dilution series.
4. Validate inter-assay reproducibility across independent automated runs.

The project was structured in two phases using the CyBio FeliX liquid handler with integrated Pan AAV Capsid Titer Assay analytics:

Phase 1: Format and Equipment Validation

• Compare dynamic range across 96-well and 384-well plate formats.
• Evaluate plate reader configurations with and without aperture spoon.
• Assess luminescence crosstalk in opaque 384 well plates at three spatial well positions relative to a high-signal positive control well.

Phase 2: Automated Reproducibility Assessment

• Use the CyBio FeliX to transfer 96 clarified HEK293 lysate samples into 384-well assay plates across three independent runs, each with an independently prepared standard curve.
• Calculate per-well CV across all three runs to determine inter-assay precision.

Our Solution

The Pan AAV Capsid Titer Assay uses Lumit® immunoassay technology built around a split NanoLuc® luciferase system. An anti-pan AAV antibody is individually labeled with two complementary luciferase subunits, LgBiT and SmBiT. When both bind a shared conformational epitope on intact capsids, the reconstituted NanoLuc® enzyme generates a luminescent signal proportional to intact capsid concentration. 

Because the epitope is conserved across serotypes, a single formulation covers both common and novel engineered variants without protocol adjustments. The no-wash, add-and-read workflow delivers results in under one hour. The CyBio FeliX liquid handler was deployed to transfer samples from a 96-well source plate into 384-well assay plates, processing 96 unique samples alongside an independent standard curve per run.

The Results

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Key performance highlights include:

• Maintained Dynamic Range: No significant difference between 96-well and 384-well formats, enabling confident miniaturization for high-throughput screening.
• Crosstalk Fully Resolved: Complete elimination of luminescence crosstalk in opaque 384-well plates using an aperture-equipped plate reader.
• 11.4% Mean CV: Strong inter-assay reproducibility across three fully independent automated runs (n =96 samples per run).
• Broad Serotype Coverage: Consistent linear performance (R² >0.99) across eight serotypes, including novel engineered variants AAV-PHP.eb and AAV2.7m8.
• Rapid No-Wash Workflow: Results in under one hour with no sample purification, integrating directly into automated liquid handling platforms.

Future Outlook

Multus plans to integrate the Pan AAV Capsid Titer Assay directly into its MediOP™ platform for AAV manufacturing media optimization, enabling systematic formulation screening across multiple serotypes and cell line backgrounds. The assay's pan-serotype coverage makes it well-suited to keep pace with the continued emergence of novel engineered capsid variants. 

By combining PROGEN's Pan AAV Capsid Titer Assay with MediOP™'s automated experimentation capabilities, AAV developers gain a scalable, serotype-agnostic titer workflow supporting both early-stage process development and commercial media optimization.

Technical Note: Capsid titer quantification utilized PROGEN's Pan AAV Capsid Titer Assay employing Lumit® immunoassay technology and NanoLuc® split luciferase complementation (Promega Corporation). Automated liquid handling was performed using the CyBio FeliX liquid handler (Analytik Jena, an Endress+Hauser Company). Independent external validation was conducted by six laboratories across multiple serotypes, including common and custom capsids.

See here for more information: Pan AAV Capsid Titer Assay

Founded in Heidelberg, Germany, in 1983, PROGEN provides labs worldwide with reliable, user-friendly, and high-quality analytical tools for gene therapy development. PROGEN’s portfolio spans AAV assays, antibodies, LVV quantification assay, and LNP kits, combining decades of expertise with a collaborative approach to help scientists deliver safe, scalable, and life-changing therapies. Visit the PROGEN website for more information.

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