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CE and HPLC applications for rAAV characterisation

Despite recent advances in the gene therapy field, significant challenges lie ahead for the consistent manufacturing and release of these medicines. Analytical methods and automation need to keep pace as more medicines move towards the market and more quality control resources are required. Well-developed methods on robust platforms will be crucial to ensure the quality of these novel medicines for the doctors and patients who need them.

At Gyroscope Therapeutics we strive to identify technologies that can transform gene therapy development and manufacturing, as well as how we release these medicines for patients in time. The examples below offer a glimpse into what is happening in our development labs to achieve these goals.

Vector analysis by CE-SDS

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is the traditional method for confirming the identity of recombinant adeno-associated virus (rAAV) vector material and analysis of vector purity. It is a well-established approach which has been used as a QC tool since the dawn of AAV vectors. However, SDS-PAGE suffers from poor resolution, is susceptible to operator to operator variations, and lacks robust quantitation of detected bands.

A capillary electrophoresis-sodium dodecyl sulphate (CE-SDS) method offers a state-of-the-art approach to characterise vector preparations. It addresses all the weaknesses of SDS-PAGE and, with the right sample preparation, requires less sample volume whilst improving the signal to noise ratio. Overall, CE-SDS is a higher resolution technology than SDS-PAGE, therefore ticking all the boxes needed for a robust product release method.

At Gyroscope we use CE-SDS with laser-induced fluorescence detection for the purity analysis and identity confirmation of our rAAV vector materials. We have adapted the protocol recently published by SCIEX [1] and found both the sample preparation as well as the data analysis straightforward. Figure 1 below shows a dilution series of the same sample and illustrates good reproducibility across the three VP peaks.

Figure 1 – Serial dilution of AAV vector analysed by CE-SDS

A more significant benefit of CE-SDS over SDS-PAGE is automation and more streamlined data analysis. Although it is possible to run multiple SDS-PAGE gels in parallel, the setup is time-consuming. While the analysis of a single sample on CE-SDS also requires 30 minutes the instrument can be left to run overnight.

This results in more data than would be provided by SDS-PAGE. Data analysis on the next day is also easier to automate on a CE instrument. All raw data is available in digital format and compared to the multiple steps required by SDS-PAGE, the final result on CE is only a mouse click away.

However, nothing comes without a price. For CE this is the upfront investment in the equipment. In addition, there is the element of time and resources required to become familiar with a new instrument and control software, as this platform is typically not being used in a standard molecular biological lab.

Long term this investment will pay its dividend by the quality of assays being available for product characterisation. After all, CE-SDS is not the only CE method that can be applied to rAAV products.

Using HLPC for vector titration

By far the most prominent platforms for genomic titration of rAAV vectors are based on quantitative polymerase chain reaction (qPCR). The move of digital PCR (dPCR) into rAAV testing has significantly improved the accuracy and precision of sample titres.

Nevertheless, qPCR based approaches are still hampered by time-consuming sample preparations which limit throughput and increase time to result. This is because multiple pre-treatment steps are required to reliably remove residual contaminating DNA which could lead to an overestimation of the sample titre and break up vector particles to release the encapsidated DNA. Similarly, immunoassays for capsid titration require multiple plate handling steps and incubations which bind resources.

We have therefore started to evaluate chromatography-based approaches as orthogonal methods for these titration assays in Gyroscope. The inspiration for using high-performance liquid chromatography (HPLC) has come from a couple of assays that have been published in recent years combined with our in-house expertise in developing the preparative chromatography steps.

This has resulted in a set of powerful analytical tools to characterise rAAV material. To stay with the genomic titre assay, we’re using ion-exchange chromatography to titre our vectors in a fraction of the time it takes by qPCR/dPCR. We then confirm interesting observations by qPCR/dPCR. We found this to be an approach with huge potential for development programmes: screening the samples on HPLC, and then re-running those of interest in a qPCR-based assay.

So far, the correlation between genomic titre obtained by the two platforms is high (see Figure 2 below). However, more work is required in understanding the assay performance in the long run and understanding how varying quality of samples may impact HPLC results. It’s a small step applying the chromatography principles from preparative to analytical scale but will be a giant leap for HPLC-based vector analytics once these methods have been shaken down and have demonstrated their feasibility under routine manufacturing conditions.

Figure 2 Correlation of genomic titre obtained by dPCR vs ion-exchange HPLC


Implementing these new platforms requires both well-characterised material and established procedures for testing the same attribute. For this purpose, orthogonal methods are an analytical scientist’s best friend. Whilst we do want to take advantage of the state-of-the-art platform and the higher throughput they offer, we acknowledge that by operating under R&D conditions none of our assays are formally validated. Confirmation of results on orthogonal methods is, therefore, a crucial activity in the development process.

Franz Schnetzinger is Director of Quality Control and CMC Analytical Development at Gyroscope Therapeutics.

The 3rd Global Bioprocessing, Bioanalytics and ATMP Manufacturing Congress will take place in Dublin, Ireland this September. Download the agenda to see what the 30+ presentations will cover.


[1] Tingting Li, Handy Yowanto, Sahana Mollah (2019) Purity Analysis of Adeno-Associated Virus (AAV) Capsid Proteins using CE-LIF Technology.

[2] SCIEX. (last accessed 7-Jan-2020)

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