GEN-MKT-18-7897-A
Mar 12, 2025 | Biopharma, BioPhase 8800 system, Blogs | 0 comments
Read time: 10 minutes
In a recent webinar, available on demand, the scientists Luiza Chrojan and Ryan Hylands from Pharmaron, provided insights into the deployment of capillary gel electrophoresis (CGE) within cell and gene therapy. Luiza and Ryan shared purity data on plasmids used for adeno-associated virus (AAV) manufacturing and data on AAV genome integrity, viral protein (VP) purity and VP ratios using the BioPhase 8800 system.
In this blog, I summarized Luiza’s and Ryan’s answers to the most pressing questions during the Q&A. Check out the Cell and Gene Therapy Compendium or contact us for further information.
Is system optimization required when transferring methods from the PA 800 plus system to the BioPhase 8800 system?
Luiza Chrojan: Most methods should be directly transferable however, we have noticed a couple of slight differences. For instance, for the sample injection, you can achieve a minimum of 0.3 psi on the BioPhase 8800 system, whereas on the single capillary PA 800 Plus system, you can go slightly lower to as low as 0.1 psi. That is something to consider if you work with low-pressure injections and want to transfer methods between system types although you should be able to get comparable data across the two platforms. Another slight difference is the sample vials available. For the BioPhase 8800 system, there is one plate type currently available, which requires a minimum of 50 microliters (µL) per sample. For the PA 800 Plus system, a low sample volume vial—the NanoVial—is available. You can use as low as 5 µL of sample. In addition, you can use regular vials, PCR Microvial inserts, and 96 well plates. Lastly, while both systems are compatible with the Empower Chromatography Data System (CDS) from Waters, the user interface differs. The BioPhase 8800 system leverages the BioPhase software interface, whereas the PA 800 Plus system uses the Empower CDS interface from Waters. Although there are some slight differences between the two systems, comparable data should be achieved as we have been able to demonstrate with transferring the AAV purity assay from the PA 800 plus system to the BioPhase 8800 system.
Did you characterize impurity peaks in your genome integrity assay?
Ryan Hylands: It is quite a challenging question and area. There are a few things we were looking at to characterize the peaks, and I touched on that in the webinar. As a first step, you can aim to identify the nature of the impurity peaks: whether they are genome-related impurities, such as genome fragments, or whether they are other nucleic acid impurities, such as host cell DNA (hcDNA), which can either be encapsidated or present in low amounts within the AAV sample. We used fractions derived from a chromatography based empty:full separation step to compare the peak profiles using CE. The empty AAV fraction should essentially contain no genome. You can compare the peak profile of that sample with the full capsid to identify common peaks. Any peaks that appear in the empty capsid control sample will indicate that they are non-genome related. Peaks only present in the full AAV capsid sample can be attributed to the intact genome or genome-related impurities.
Another approach is to pretreat the sample with nucleases like benzonase to digest nucleic acids that are not encapsidated. Clean-up of the pre-treated samples before denaturation of the capsids is crucial to avoid accidental genome digestion. A comparison of the peak profiles of treated and untreated samples provides information on encapsidated vs. non-encapsidated impurities.
I want to highlight that we estimate the size of the impurities by comparing them with the single-stranded size ladder. This can help identify them.
Another approach could be to use restriction enzyme digestion based on restriction sites in your genome. If an impurity peak disappears after the treatment with a restriction enzyme, it indicates that the peak likely contained the digestion motif sequence and was related to the genome.
How well does your CE data correlate with other empty/full assays, such as analytical ultracentrifugation (AUC)?
We are currently running a study comparing various assays used to monitor empty, partially filled, and full capsids. The CE assay we presented is among those assays. I see CE as an orthogonal technique to analytical ultracentrifugation (AUC). The CE genomic integrity assay shows you what is inside the capsids, if the genome is intact, and the abundance of the intact genome. AUC measures the sedimentation coefficients of viral particles, AAV with packaged genomes have higher sedimentation coefficients that those without. The output is a relative quantification of empty, partially filled, and full capsids with the limitation of not being able to determine whether a single genome, or multiple fragments with the same total mass are present. The synergy of the two techniques allows us to look at both of these aspects, and to provide a more complete understanding of the product. We are actively building up that dataset the more samples we test.
How do you set up a system suitability test or reference standard injections on the multi-capillary BioPhase 8800 system?
Luiza Chrojan: We also asked ourselves that same question when we began working with the BioPhase 8800 system since it differs from how we set up the single capillary system. On the multi-capillary system, you inject eight samples at once. Currently, we analyze a reference standard sample across different capillaries at the start of the sequence as an SST and bracket the sample injections throughout the sequence. This ensures the data is comparable and consistent across the eight capillaries and throughout the sequence.
Using the CE-SDS purity method, how do you calculate the VP ratio?
Ryan Hylands: The VP ratio is the abundance ratio of VP1, VP2, and VP3. Theoretically, a 1:1:10 ratio is expected, with VP3 being the most abundant. Once we separate the proteins in the electropherogram using CE-SDS, we can take the peak areas and determine a ratio against VP1. Because we are using fluorescence dye labeling, there is something to consider: We use a Chromeo P503 dye, which is also mentioned in technical notes from SCIEX, for labeling viral proteins. This dye binds to the proteins’ primary amines, so primarily lysines. At Pharmaron, we have devised a method in conjunctions with Empower CDS from Waters that takes this into account and automatically generates a more accurate calculation of the VP ratios. We have seen a growing interest and requirement from clients to use the CE-SDS to infer correct formation of capsids.
Why is the peak migration order changing when using the new DNA 20 kb Plasmid and Linear kit compared to the dsDNA 1000 kit?
Luiza Chrojan: Indeed, the peak migration order differs between the two kits. The difference is due to the fluorescent dyes used in the two kits. With the dsDNA 1000 kit, we use the LIFluor EnhanCE dye. However, with the new DNA 20 kb Plasmid and Linear kit, we used the SYBR™ Gold Nucleic Acid gel stain1.
The two dyes will impact the confirmation and the charge-to-size ratio of the plasmid isoforms. That results in a change in migration order. With the LIFluor EnhanCE dye, we see the linear isoform first, then the supercoiled, and then the nicked/open circular. With the SYBR™ Gold Nucleic Acid gel stain1, the supercoiled isoform migrates first, then the linear and the open-circular last. As shown in our slides, you can do a restriction digest to confirm the isoform species. Also, the plasmid standard provided in the DNA 20 kb Plasmid and Linear kit contains the main three isoforms, which helps identify the migration order of your peaks.
You showed that in-process plasmid samples can be analyzed. Can you also analyze AAV in-process samples with the CE assays you presented?
Luiza Chrojan: We can assess in-process AAV samples. We analyze samples from the capture chromatography step onwards. While we use an electrokinetic injection, which would typically be affected by high salt concentrations in the sample or formulation buffer, we can achieve comparable responses with the in-process samples compared to purified material using the optimized CE methods without any additional modifications. We can assess the purity of AAV samples across the process with CE and provide end-to-end support.
Is there a plasmid purity specification that you target? And what happens if your target purity is not met?
Ryan Hylands: Yes, we have a target specification for the percentage of supercoiled plasmid, which is set based on regulatory guidelines for plasmid, along with batch performance data. The target specification is commonly 85% or greater. We typically achieve purities well above 90% with bulk material and meet the target specification. In the unlikely event that we didn’t meet that specification and the purity assays were valid—meaning all the standards and controls within the assay passed—it would prompt us to look back at other aspects of that process. We would examine if the process went according to plan and if, for example, the chromatography steps worked as expected to achieve the desired purification. We would consider the test results of other assays to understand if the impurity profiles changed, for instance, due to a process change.
Which AAV serotypes did you analyze with CE-SDS-LIF?
Ryan Hylands: We have looked at multiple AAV serotypes, and I showed a few in my slides. For example, we analyzed AAV2, AAV5, AAV8, and AAV9, which are common serotypes used in drug development. We use a platform CE-SDS-LIF assay for all serotypes. The only difference is the sample preparation which is optimized for each of the serotypes to minimize any sample preparation-induced fragmentation.
For plasmid in-process analytics, do you see other impurities in the samples, such as hcDNA?
Luiza Chrojan: Yes, this is possible. We analyze plasmid cell paste or harvest material and the purity following the upstream batch to understand the supercoiled content at these stages. During method development, we observed an unknown peak in these samples and suspected it was related to nucleic acids. After a thorough investigation, we were able to characterize the unknown peak and confirm the presence of host cell DNA. There is a possibility that we could see host cell RNA (hcRNA) with the same assay. However, we have a very stringent process to remove hcRNA and have another assay to test specifically for hcRNA.
What sample concentrations are required for the AAV CE-SDS-LIF purity method?
Ryan Hylands: It depends on the serotype since their response rate behaves differently in assays. AAV2 can require a higher concentration to achieve a similar response compared to other serotypes. For AAV5, for example, we can achieve a good signal for samples with a lower concentration.
Did you assess the identity of any peaks detected in the empty AAV fraction of your genome integrity assay?
Luiza Chrojan: As part of our studies, we are currently looking at orthogonal assays for genome integrity with CE. We expect to have an update on this topic in 2025.
Can you expand the CE-SDS-LIF assay to analyze other viral vectors, such as lentivirus or adenovirus?
Ryan Hylands: We typically use CE-SDS-LIF for analysis of AAV but do get requests for this type of work. SCIEX analyzed lentivirus samples, which are published in this technical note. For other viral vectors, I suggest contacting SCIEX to learn more about the applicability of the assays.
1SYBR™ is a trademark of the Life Technologies Corporation. SYBR™ Gold Nucleic Acid gel stain is not available for resale.
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