How to achieve fast, accurate and reliable quantification of AAV serotypes (2023)

Content sponsored byGator Bio, Inc.November 19, 2021Reviewed by Alex Smith

perceptionsof the industryIndrani Chakrabortysenior scientistGator Bio, Inc.

In this interview, News-Medical speaks with GatorBio's Indrani Chakraborty about how they achieved accurate and reliable quantification of AAV serotypes.

Strategies for accurate, easy, and rapid quantification of AAV serotypes

Video credit: Gator Bio

Can you give us an overview of Gator Bio and the work he's done with Biolayer Interferometry (BLI)?

Gator Bio has worked with BLI for nearly two decades. Hong Tan, the inventor of biolayer interferometry, founded FortéBio in 2001. Subsequently, a prototype BLI instrument was developed in 2002, which was released in 2006 as Octet.

Hong Tan continued his work with Bob Zuk and developed the BLI technique to overcome the limitations of the first generation of BLI instruments. This, in turn, led to the development of the next generation of BLI instruments, which led to the founding of Gator Bio by Hong Tan and Bob Zuk in 2018.

Since then, two new BLI systems have been released: GatorPrime and GatorPlus. The first is a basic system that supports two 96-well plates, while the second is a higher throughput system that supports both 96- and 384-well plate formats.

As a company, Gator Bio is in a healthy state both financially with over $100 million in funding and intellectual property with over 50 patents already granted.

Our biological layer interferometry technology was originally launched on a pylon platform for diagnostic purposes. It was initially available through our sister company ET Healthcare for diagnostics in China, resulting in the installation of over 2,000 systems across China.

In the US, the same core technology will be integrated into the GatorPrime and GatorPlus research exploration systems. These instruments have sold well over the past two years and sales continue to show rapid growth.

The biosensors for the BLI instrument are manufactured in Palo Alto, California, where our main research and development efforts are also located.

Our team of experienced engineers and field scientists help customers use their Gator Bio products. This team of experts is located in the United States and teaches customers how to succeed with our platforms.

We support three core applications consistent with the capabilities of biological layer interferometry technology: quantification, kinetics, and epitope categorization.

precision and reproducibility: Serotype AAV5 was used to test accuracy and reproducibility. The table above shows a recovery close to 100% and a CV ranging from approximately 1.5% in medium and high titles to 10.9% in low titles. Photo credit: GatorBio

Where do BLI platforms find their best fit in the biotherapeutic discovery pipeline?

Our BLI platforms fit well throughout the biotherapeutic discovery process, from initial discovery to user executionantibody titerMeasurements for early development, where users can perform epitope binning.

Gator platforms are advantageous when it comes to analyzing lead antibodies and understanding binding kinetics. They are also useful further down the process scale where they can be used for quantification.

Gator platforms remain useful after the discovery process as they are also useful in CMC, manufacturing and QC applications where they can be used to perform kinetic measurements.

Gator Bio's two platforms, GatorPrime and GatorPlus, differ mainly in their performance. A wide range of biosensors and consumables are available, as well as a wide range of application-specific software that includes tools for areas such as data acquisition, analysis and reporting of single and dual plate operations on both Gator Bio platforms.

Gator Bio can also provide on-demand and custom probes, which means our instruments and tools can be used in any number of environments.

What can you tell us about the new AAVX biosensor and how it fits into the BLI space?

The AAVX biosensor was released on September 15^º, 2021, and we are very happy with the response. We were impressed with the interest and shipment of the sensor and systems to new customers. So far, our biosensors and technologies have mainly focused on the antibody, small molecule drug and stimulant drug markets. Recently, we've started to look more closely at the areas of AAV and gene therapy.

As with many other biophysical techniques, biological layer interferometry (BLI) is part of a label-free system. In this case, label-free means you don't need to use a special dye or tag designed to be conjugated to a molecule of interest, allowing you to observe binding via fluorescence or illumination. Instead, BLI uses white light.

This robust bioanalytical technique has been used in a variety of laboratories and applications for nearly 17 years. However, this is probably the first direct application of a tag-free biosensor in gene therapy, and in particular for AAV.

Gator Bio's BLI instruments consist of three main components: the motor, the biosensor, and the software. The biosensor is where all the reactions take place, particularly where the white light passes through the two ends of the tip.

The biosensor comprises two layers, the optical layer and the surface layer. While the optical layer remains constant, the surface layer is modified and we can actually make adjustments to the surface layer to make specialized probes.

In the case of the AAVX probe, we use an industry gold standard, CaptureSelect^MTThermo Fisher camelid antibody that binds to AAV serotypes 1-8 and 10.

When light falls, it reflects off these two surfaces. In cases where something binds to the probe, there are observable changes in the light reflection index - a nanoscale change is visible over time. This is registered with the instrument software.

When light and binding is complete, you can dip the probe into another solution containing an analyte of interest that will bind to it. This method can be used to perform kinetic experiments to calculate the activation rate.

As you move the probe into the buffer, any bound analytes slowly dissociate, and watching this dissociation in real time provides the off rate.

Once the activation and deactivation rates are determined, it is possible to calculate the Kb or binding constant.

The AAVX probe can be used in addition to quantification to observe direct binding to the biosensor surface. It can also be used in kinetic experiments to determine the rate of binding activation and deactivation.

Binding rate of different AAV serotypes: The binding rate of 7 different AAV serotypes was examined. The figure below shows the binding rates of serotypes tested in 2 x 10¹¹concentration vp/ml. Photo credit: GatorBio

What are the benefits of GatorPrime and GatorPlus instruments and how do they work in practice?

We currently offer two models of BLI instruments: GatorPrime and GatorPlus. Each instrument can contain two plates, one plate is for the biosensor, which is kept and hydrated in buffer, and the remaining plate is the reaction plate.

Experiments can be set up on the reaction plate via a software interface. A series of wheels can be used to apply a series of biosensors to the plate.

This system offers a number of advantages. For example, there are no fluids involved and there are no channels or flow cells in which the plug is transported. The lack of fluids allows for easy maintenance and there is little chance of the instrument becoming blocked during experiments.

The easy-to-use software is also an important benefit. The other notable advantage of this technique is its ability to switch the biosensor from one row to another between samples on the plate.

As the sample does not need to be mixed or added, valuable samples remain intact on the plate, useful in cases where additional characterization may be required.

Due to the lack of fluids in the system, cell supernatants, cell lysates and plasma can also be used in the assay plate. Due to the variety of biosensor types offered, a wide variety of samples can be accommodated, and we can also create a custom biosensor designed specifically to meet each customer's needs.

Are there specific challenges when working with AAV quantification and how can BLI help address these challenges?

In the past year, we have built a large portfolio of biosensors for antibody drug discovery. Our goal was to advance into new therapeutic areas with our existing biosensor in order to make BLI technology accessible to more researchers.

When we started thinking about gene therapy applications, AAV was certainly the first thing that caught our attention.

We begin by examining the analysis techniques that researchers are currently using in their laboratories for AAV development. We discovered an article that highlighted the extensive use of ELISA for total AAV quantitation, along with the use of GDPCR and AUC in the characterization of complete CAP MTN sets.

Our research found that the analytical techniques used in most other drug discovery processes allowed for greater throughput than those used in AAV, and we concluded that the AAV process is currently slower due to the low throughput of analytical techniques and the very long processing time. long for researchers.

These issues can lead to loss of sampling, aggregation, and other issues from the upstream process to the downstream process. Therefore, our goal was to use BLI technology to improve throughput capabilities inAAV applicationsand allow experiments to be carried out as easily as possible.

ELISA was a technique that could easily be converted to BLI, so we started there.

An ELISA technique usually involves a series of steps. First, researchers need to prepare the plate and then add the sample that will bind to the plate (possibly an immobilized antibody).

After several washes, a detection antibody is added before incubating for a period of time. The last step involves adding a substrate and the sample is cooled and read.

This is a time consuming process whereas when using the BLI all you need to do is dip the pre-coated custom biosensor into the desired solution. This then turns on and you can watch the nanometer shift change over time, calculate the rate of degradation, and calculate the concentration. This is a very simplified process compared to the traditional ELISA technique.

When we examine the ELISA kit, we find that the linear curve fit of the ELISA concentration range is generally between E⁶Toe⁷particles per milliliter, which is much smaller than most samples researchers want to work with.

This means that we usually have to make several dilutions from the working concentration to get within the ELISA range. However, this process can lead to errors.

The goal of our AAVX probes was to create a tool that could capture the spectrum that researchers want, but without the need for manual dilution.

The AAV quantification process depends on the rate of binding of the AAV serotype to the probes on the biosensor platform. We used the commonly used CaptureSelect AAVX antibody courtesy of a contract with its manufacturer. This antibody is commonly used in AAV purification, part of the Thermo Fisher affinity column.

As a result, as the AAVX probes the column, it can identify all serotypes – AAV1-8 and 10. This offers an additional benefit as researchers working with AAV2 and AAV5 do not need to purchase two different ELISA kits, reducing costs.

Photo credit: GatorBio

Can you tell us about the relationship between serotype, concentration and binding rate and how the AAVX CaptureSelect antibody can help clarify this relationship?

The rate of binding depends on concentration and serotype. Several published papers have identified residues in the AAVX antibody related to AAVX serotypes.

These studies also showed that AAV1, unlike AAV8, binds to the AAVX CaptureSelect antibody. These binding differences resulted in different binding rates, which in turn resulted in different signal strengths.

As with the ELISA method, determination of an unknown concentration requires construction of a standard curve, which the BLI can readily facilitate. Running through this entire 96-well plate can be done in about 26 minutes, assuming each plate would take about 2 minutes for a given concentration range.

This speed is achieved because no wash tests, dilutions or substrate additions are required. Researchers can simply pipette the solutions of interest into the plate and click Start - everything from there is automated and there are few touchpoints.

Our AAVX biosensors are reusable, what we call regeneration, which we achieve by attaching an anti-AAVX ligand to the probe. This interaction is covalent and unbreakable, unlike ligand binding to the AAV serotype, which can be easily broken.

Researchers can elute AAV from the column by simply passing it through an acidic solution or regeneration buffer to release the AAV. With this approach, the biosensor can be reused multiple times – at least 10, but possibly more.

How do concentration ranges and binding rates affect AAV characterization ability?

When examining our instrument's ability to quantify AAV, it's important to keep in mind that we don't have our own AAV to characterize - we buy it from vendors. Consequently, the largest concentration we have managed to buy so far has been E¹³Pro milliliter particles.

A customer wanted to test our probes and extend the concentration limit because they concentrate more in the highest concentration regions. This client was looking for a job at E¹⁴Particle concentrations per milliliter - an achievable level.

We characterized all standard AAV serotypes from AAV1-8 and 10 and also evaluated our probes to examine the binding rate for each serotype. Characterization and evaluation were performed to confirm that the probes function in the high, medium and low concentration ranges for each AAV serotype.

The CaptureSelect AAVX antibody has been found to bind to nearly all serotypes, albeit at slightly different residues due to different binding rates.

Binding rate is always temperature dependent. Although experiments are normally performed at room temperature, it is possible to configure the instrument for specific temperatures up to 40 degrees.

While this process can be complex, standard curves do not need to be run repeatedly. The curve is executed at the beginning of the process and the software saves this standard curve. Whenever an unknown curve is run, it is possible to recall the standard curve and calculate the concentration from that standard curve.

For individual serotypes it is essential to maintain an individual standard curve. Because our probes work flawlessly in the high, medium, and low concentration range and allow for good accuracy and recovery when performing AAV purification, you can use them across the full range of an assay and with almost any type of media.

The software was able to efficiently provide the correlation across the various samples we evaluated. When we analyzed an AAV2 standard curve and an unknown curve to run our AAV-like probes and ELISA side-by-side, we found that the agreement between the AAVX probe for Gator and the current ELISA was exceptional.

It is important to consider that ELISA kits use different antibodies for different serotypes. Therefore, if you are considering switching from ELISA to BLI, it is important to check compatibility.

We have found that most users are comfortable working with AAV concentrations of E⁹Toe¹³. Our probes work well in this specific concentration range without the need for dilution, and because our system has eight channels running simultaneously, throughput is high regardless of concentration.

Our customers have taken our probes to the limit and some have already used them for E¹⁴particles per milliliter. I anticipate that this will be the case with the development of new assays and that the boundaries can be expanded further.

What are Gator Bio's next steps in terms of developing or expanding instrument capabilities?

BLI is the first unlabeled product to enter the gene therapy market, so we are evaluating several biophysical assays and techniques that we could implement on our BLI platform.

One customer is already using our probes to load AAV serotypes and study antibody binding and dissociation association to measure and study kinetics.

When using the AAVX probe, it is also possible to use any commercially available reagent to develop your own AAV assay. You can take advantage of this with our assay biosensors and use them as a kind of custom-made biosensor.

As we also offer mouse, MFc and HFC probes, researchers who want a specific biosensor for a particular antibody have this option. It is possible to create any type of probe to perform any type of experiment, as the system was designed to be very open.

We encourage users to research new ways to use the probe, as it has great potential in a variety of applications. We make the product and we love helping users take it to the next level, realizing its full potential.

About Indrani Chakraborty

Indrani Chakraborty has more than 17 years of research experience in chemical and physical characterization of biological products in biopharmaceutical companies such as BMS, Medarex, Scios, etc. Indrani's research areas include biosensors and surface plasma resonance platforms for characterization ofAntigen& Antibody Binding Kinetics, Termodinâmica, Epitope Binning, Antibody Stability and Developability, Biologics Formulation and Fcgamma Receptor Binding Studies.

About Gator Bio, Inc.

Gator Bio is a leading global biosensor company headquartered in Palo Alto, California. At theGator Bio,We provide researchers with the tools and instruments to advance their research. From antibody development to small molecule discovery to basic research, Gator Bio can be used to give meaning to the unknown. From the original inventors of Label-Free Biolayer Interferometry (BLI), Gator Bio delivers the next generation of BLI technology.

Forms:

• Rapid quantification of unknown samples (raw or purified)
• Epitop-Binning
• Kinetik Tracking/Sorting out of rate
• Affinity determination (pM ​​to mM)
• Antibody isotyping/subtyping

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