How to achieve fast, accurate and reliable quantitation of AAV serotypes

insights from industryIndrani ChakrabortySenior ScientistGator Bio, Inc.

In this interview, News-Medical talks to Indrani Chakraborty from GatorBio about how they have achieved accurate and reliable quantitation of AAV serotypes.

Strategies for Accurate, Easy and Fast Quantitation of AAV Serotypes

Video credit: Gator Bio

Could you give us an overview of Gator Bio and the work it’s been doing with biolayer interferometry (BLI)?

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

Hong Tan, along with Bob Zuk, carried on with their work, developing the BLI technique to address the restrictions of the first generation of BLI instruments. This, in turn, resulted in the next generation of BLI instruments being developed, leading to the formation of Gator Bio by Hong Tan and Bob Zuk in 2018.

Since then, two new BLI systems have been brought to the market: GatorPrime and GatorPlus. The first one is an entry-level 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 finds itself in a healthy state, both financially with over $100 million in funding, and in relation to IP, with over 50 patents already granted.

Our biolayer interferometry technology initially came to market for diagnostic purposes on a Pylon platform. It was initially available for diagnostics in China via our sister company ET Healthcare, leading to over 2,000 systems being installed throughout China.

In the United States the same core technology is incorporated in GatorPrime and GatorPlus research use systems. These instruments have sold well in the last two years, and sales continue to display rapid growth.

The biosensors for the BLI instrument are manufactured in Palo Alto, California, which is also the location of our key R&D efforts.

Our team of experienced engineers and field scientists support customers when using their Gator Bio products. This team of experts is located across the United States and is teaching customers how to be successful with our platforms.

We support three core applications in line with the abilities of biolayer interferometry technology: Quantitation, Kinetics, and Epitope Binning.

Accuracy and reproducibility: AAV5 serotype was used to test the accuracy and reproducibility. The table above shows recovery close to 100% and CV ranging from around 1.5% at medium and high titers to 10.9% at low titer. Image credit: GatorBio

When it comes to the biotherapeutics discovery pipeline, where do BLI platforms find their best fit?

Our BLI platforms fit well across the complete biotherapeutics discovery process, from early discovery, where users can carry out antibody titer measurements right through to early development, where users can carry out epitope binning.

The Gator platforms are beneficial when it comes to analyzing lead antibodies and understanding binding kinetics. They are also of use further downstream in process scale-up, where they can be utilized for quantitation.

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

Gator Bio’s two platforms, GatorPrime and GatorPlus, are differentiated primarily on the basis of their throughput. A large array of biosensors and consumables are available, in addition to a wide range of application-specific software incorporating tools for areas such as data acquisition, analysis and reporting of one or two plate operations on both Gator Bio platforms.

Gator Bio can also supply on-demand probes and customized probes, meaning our instruments and tools can be utilized in any number of settings.

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

The AAVX biosensor launched on September 15th, 2021, and we’re really excited about its reception. We are overwhelmed with the interest and shipping the sensor and systems to new customers. Up to now, our biosensors and technology have mainly focused on the antibody, small molecule drug discovery, and pep drug discovery markets. Recently, we began to explore the AAV and gene therapy fields in greater detail.

As with several other biophysical techniques, biolayer interferometry (BLI) is part of a label-free system. In this instance, label-free means that you do not have to employ a dye or special tag designed to conjugate to a molecule of interest so you can observe binding via fluorescence or illuminance. BLI utilizes white light instead.

This hardy bioanalytical technique has been utilized in a range of labs and applications for almost 17 years. However, this is likely the first direct application of a label-free biosensor when it comes to gene therapy, and especially for AAV.

Gator Bio’s  BLI instruments have three main components: the machine, the biosensor and the software. The biosensor is the location where all the reactions take place, primarily where the white light passes through on both 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 undergoes modification, and we can, in fact, carry out surface layer customization to create specialized probes.

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

When the light is incident, it is bounced back from these two surfaces. In instances where anything binds to the probe, there are observable changes in the light’s reflective index - a nanometer shift is visible over time. This is recorded utilizing the instrument’s software.

When the light and binding have been completed, you can dip the probe into another solution containing an analyte of your interest which will bind on top of it. This process can be used for performing kinetic experiments to calculate the on-rate.

By moving the probe into the buffer, any bound analytes will slowly dissociate, and observation of this dissociation in real-time will give you the off-rate.

When the on-rate and off-rate have been ascertained, it is then possible to work out the Kb or the binding constant.

The AAVX probe can be additionally utilized for quantitation to observe direct binding to the surface of the biosensor. It can also be employed in kinetic experiments to determine the on-rate and off-rate of the binding.

Binding rate of different AAV serotypes: The binding rate of 7 different serotypes of AAV were studied. The figure below shows binding rates of tested serotypes at 2 x 1011 vp/mL concentration. Image credit: GatorBio

What benefits do GatorPrime and GatorPlus instruments offer, and how do these operate in practice?

At present, we offer two models of BLI instruments: GatorPrime and GatorPlus. Each instrument can hold two plates, where one plate is for the biosensor, which is kept and hydrated in the buffer, and the remaining plate is the reaction plate.

Experiments can be set up in the reaction plate by means of a software interface. A series of wheels can be utilized to apply a range of biosensors to the plate.

This system provides a number of advantages. For example, no fluidics are involved, and there are no channel or flow cells where the buffer is transported. The absence of fluidics allows for easy maintenance, and there is little likelihood of the instrument becoming blocked during experiments.

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

As there is no need to mix or add things to the sample, valuable samples are left intact on the plate, which is useful in instances where there may be a need for further characterization.

The fluidics-free nature of the system means it is also possible to use cell supernatant, cell lysates and plasma in the assay plate. A variety of samples can be accommodated due to the range of biosensors types on offer, and we can also create a customized biosensor that is designed especially for the individual customers’ needs.

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

In the past year, we have established a large biosensor portfolio meant for antibody drug discovery. We were aiming at venturing into new therapeutics areas with our existing biosensor to make BLI technology accessible to more researchers.

When we began to think about gene therapy applications, AAV was certainly the first one to stand out.

We started examining the analytical techniques that the researchers were currently using in their labs for AAV development. We discovered a paper that highlighted extensive utilization of ELISA for total AAV quantitation, along with the utilization of GDPCR and AUC in characterizing MTN full CAP sets.

Our research revealed that the analytical techniques employed in the majority of other drug discovery processes allowed for greater throughput than those used in AAV, and we came to the realization that at present, the AAV process is held back by the analytical techniques’ low throughput and the very long turnaround timescale for researchers.

These issues could result in a loss of sample, aggregation and other issues from the upstream and impact right through to the downstream process. Therefore, our aim was to utilize BLI technology to enhance throughput capabilities in AAV applications and allow for experiments to be as easy to perform as possible.

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

A series of steps are generally involved in an ELISA technique. Initially, researchers first have to prepare the plate and then add the sample that binds to the plate (possibly an immobilized antibody).

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

This is a time-consuming process, whereas if you utilize BLI, all you need to do is dip the pre-coated customized biosensor in the solution of interest. This then binds, and you can observe the change in the nanometer shift over time, work out the mining rate and calculate the concentration. This is a highly simplified process compared to the traditional ELISA technique.

When we examined the ELISA kit, we found that the ELISA concentration range’s linear curve fit usually ranges between E6 to E7 particles per milliliter, which is much lower than the majority of samples researchers intend to work with.

This means we usually have to carry out several dilutions from the working concentration so as to get to the ELISA’s range. However, this process could conceivably induce error.

The aim of our AAVX probes was to develop a tool that could accommodate the desired range of researchers but without the need for manual dilution.

The AAV quantitation process is dependent on the rate of the AAV serotype binding to the probes on the biosensor platform. We utilize the commonly employed CaptureSelect AAVX antibody courtesy of an agreement with its manufacturer. This antibody is generally employed in AAV purification, a part of the Thermo Fisher affinity column.

As the AAVX probes the column, as a result, it can determine all serotypes - AAV1-8 and 10. This gives an added advantage in that researchers working with AAV2 and AAV5 do not need to buy two different ELISA kits, thus reducing costs.

Image credit: GatorBio

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

The binding rate is contingent on the concentration and the serotype. A number of published papers have identified the residues on the AAVX antibody in relation to the AAVX serotypes.

These studies also indicated that AAV1 binds to the AAVX CaptureSelect antibody differently to AAV8. These differences in binding led to different binding rates, leading, in turn, to differing signal intensities.

Like the ELISA process, the determination of an unknown concentration necessitates the generation of a standard curve, which the BLI can rapidly facilitate. Running that entire 96-well plate, working on the assumption that each plate would take around 2 minutes for a particular concentration range, could be carried out in approximately 26 minutes.

This speed is achieved as there is no need for a wash test, dilutions or substrate addition. Researchers can simply pipet the solutions of interest into the plate and then hit start – everything from there on in is automated, and there are few touchpoints.

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

Researchers can elute the AAV from the column simply by passing through an acidic solution or regeneration buffer to pass out the AAV in the flow-through. Employing this approach allows the biosensor to be reused a number of times – at least 10, but possibly more.

How do the concentration ranges and binding rates impact the ability to characterize AAV?

When examining our instrument’s ability to quantitate AAV, it is vital to take into account that we do not have our own AAV to characterize - we buy this from vendors. Consequently, the highest concentration that we have been able to purchase so far has been E13 particles per milliliter.

A customer wanted to test our probes and push the concentration limit because they are more focused on the higher concentration regions. This customer was seeking to work at E14 particles per milliliter concentrations – a level that was possible to reach.

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

The AAVX antibody from CaptureSelect was found to bind to almost all the serotypes, though at slightly different residues due to the difference in their binding rates.

Binding rate is always temperature-dependent. While experiments tend to be performed at room temperature, setting the instrument to work at specific temperatures up to 40 degrees is possible.

Though this process can be complex, there is no requirement to run the standard curves repeatedly. The curve is run at the beginning of the process and the software will save that standard curve. Whenever an unknown curve is run, it is possible to recall the standard curve and to calculate the concentration against that standard curve.

For individual serotypes, it is essential to run an individual standard curve. As our probes work perfectly in the high, medium and low concentration ranges, and allow for good precision and recovery when carrying out AAV purification, it is feasible to use these for the entire range of an assay and with nearly any type of media.

The software was able to efficiently provide the correlation in a number of examples that we evaluated. When we looked at an AAV2 standard curve and an unknown curve to run our AAV-like probes and ELISA side by side, we discovered that the agreement between the AAVX probe for the Gator and the current ELISA was exceptional.

It is vital to take into account that ELISA kits utilize different antibodies for different serotypes, so if considering switching from using ELISA to BLI, it is important to double-check for compatibility.

We have found that the majority of users are focused on working with AAV concentrations from E9 to E13. Our probes work flawlessly in that specific concentration range without the need for dilution, and as our system has eight channels working concurrently, throughput is high irrespective of concentration.

Our customers have pushed our probes to their limits, and some have already used them for E14 particles per milliliter. I predict that with further assay development, and that the limits could be pushed even further.

What are the next steps for the Gator Bio in terms of developing or expanding the instruments’ capabilities?

BLI is the first label-free product in the gene therapy market, so at present, we are looking at a range of assays and biophysical techniques which we could possibly implement in our BLI platform.

A customer is already using our probes to load AAV serotypes and investigating antibody binding association and dissociation to measure and explore kinetics.

By utilizing the AAVX probe, it is also feasible to make use of any commercially available reagent to develop your own AAV assay. You can take advantage of this with our assay biosensors and essentially use this as a customized biosensor.

As we also offer Mouse, MFc and HFC probes, researchers wanting a biosensor that is specific to one particular antibody have this option. It is feasible to create any kind of probes to carry out any kind of experiment as the system is designed to be highly open-ended.

We promote users’ exploration of new ways of utilizing the probe as it has great potential in a variety of applications. We manufacture the product and are happy to support users in order to take it to the next level by exploring its full potential.

About Indrani Chakraborty

Indrani Chakraborty has over 17+ years of research experience in chemical & physical characterization of biologics at biopharma companies such as BMS, Medarex, Scios, etc. Indrani's research areas include biosensor & surface plasma resonance platforms for characterization of antigen & antibody binding kinetics, thermodynamics, epitope binning, antibody stability and developability, biologics formulation, and Fcgamma receptor binding studies.

About Gator Bio, Inc.

Gator Bio is a world-leading biosensor company headquartered in Palo Alto, CA. At Gator Bio, we provide researchers the tools and instrumentation to advance their research. From antibody engineering to small molecule drug discovery to basic research, Gator Bio can be used to bring meaning to the unknown. From the original inventors of label-free biolayer interferometry (BLI), Gator Bio provides the next generation of BLI technology.

Applications:

  • Rapid quantitation of unknown samples (crude or purified)
  • Epitope binning
  • Kinetics screening/ off-rate ranking
  • Affinity determination (pM to mM)
  • Antibody isotyping/subtyping

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