Accurate measurement of AAV genome titer throughout the development and production for gene therapy is important to ensure the effectiveness of the final product. Conventionally, this task has been accomplished using qPCR and more recently digital PCR (ddPCR) methods. Each has its own benefits and downsides.

While qPCR offers a 4 logs dynamic range, it has several drawbacks – significant variability with CV (coefficients of variation) reaching up to 30%, susceptibility to inhibitory factors such as PCR inhibitors, adverse impact of extraneous DNA, lengthy analysis times and the necessity to establish a standard curve.

The ddPCR on the other hand presents several advantages viz. it eliminates the need for a standard curve, is accurate and provides high precision ranging from 3% to 20%. However, this impressive accuracy and precision is attainable within a limited quantitative dynamic range of approx. 2logs. The ddPCR’s multiplexing capability facilitates the examination of genome integrity, thus making it very useful. That said, the ddPCR process involves extended assay times, a relatively intricate multi-step workflow, and vulnerability to PCR limitations, posing challenges for routine application. Notably, 100-to-1,000,000-fold dilution is needed for samples to fall within the working range and to minimize matrix effects seen in in-process samples, introducing error and extended sample processing time. The cost per sample can also be substantial.

The above observations motivated us to develop a fast, easy, and accurate assay for in-process samples based on a novel method that excels in precision, speed, accuracy and most importantly simplicity.

Our groundbreaking approach revolves around DNA hybridization, immunochemistry and biolayer interferometry, encompassing a streamlined two-step procedure involving lysis and hybridization within a single tube, succeeded by detection using a biosensor.

Central to the methodology are two oligo probes—an approximate 40 nt fluorescein-labelled probe and a 40 nt SuPlex probe. The SuPlex oligo includes a 30 Thymine oligonucleotide bearing biotin at both the 5′ and 3′ ends. The target region for these probes can be located within the Gene of Interest (GOI) or the promoter/enhancer regions. The lysis and hybridization steps involve heating at 95⁰ C, promptly followed by rapid cooling to 5⁰ C. The resultant hybridized sample is quantitated employing an anti-fluorescein biosensor.

The assay offers a quantitative dynamic range of 5E+9 to 5E+12 and delivers precision ranging from 3% to 9%. The assay time is mere 30 minutes. The inherent robustness of this method makes it compatible with DNase1, proteinase K, extraneous DNA, PCR inhibitors and in-process buffers, rendering it highly suitable for both research and manufacturing. Notably, another unique feature is the ability to quantify individually both positive and negative strands of the packed genome. Also, our preliminary data using oligo probes for GFP, CMV promoter, CMV enhancer and SV40 regions shows the potential for determining genome integrity.

So, while QPCR and digital PCR are currently the main tools for AAV genome titer assay, the DNA hybridization-biolayer interferometry approach described here shows significant benefits over the PCR methods. Its ability to characterize packed genome content deeper by providing strand specific titers, and robustness against various inhibitory factors positions it as an asset in both research and manufacturing contexts. Further, we have applied the same methodology for direct RNA titer without reverse transcription in Lentivirus.

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Gator Bio in partnership with GeneX India Bioscience is ready to serve the biotherapeutics research and manufacturing with next-gen biolayer interferometry

PALO ALTO, CA, UNITED STATES, April 18, 2023/EINPresswire.com/ — Gator Bio, the next-gen biolayer interferometry solutions company, has signed an agreement with GeneX India Bioscience Pvt Ltd, a Chennai-based distributor of innovative biomedical technology and products. The agreement will allow Gator Bio to expand its product offerings rapidly in India, a key region for Gator Bio’s global growth strategy.

Dr. Hong Tan, CEO of Gator Bio, said, “GeneX India has a track record of successfully introducing innovative products into market — as evidenced by its previous success in commercializing e.g., PEIpro, Double Dye Probe, FectoPRO etc. We look forward to working with the GeneX India team to bring the Gator platforms to users throughout the region, helping meet the demand for therapeutics development and manufacturing.”

“Gator BLI platforms and innovative biosensors are an important addition to GeneX India’s portfolio of Cell and Gene Therapy, Protein Biology products, and we are very excited about the possibilities the Gator shall give our customers in characterizing antibodies, AAVs and small molecule drugs,” said Mr. Felix Paul, CEO of GeneX India. “Access to this next-gen technology is key to the continued enablement of our biopharma and academia research customers in India. We are very excited about helping research and manufacturing community with the novel applications in gene therapy, immunotherapy, and lipid nanoparticle development.”

This partnership builds on Gator Bio’s commitment to providing high-quality BLI platforms globally. Gator Bio has recently entered into strategic partnerships with BMS Korea (Bio Medical Sciences), I&L Biosystems in Europe and Solve Scientific in Australia to provide sales and services to biotherapeutics and gene therapy companies. To learn more, please visit www.gatorbio.com.

About Gator Bio, Inc.
Gator Bio is a life sciences company providing bioanalytical systems to accelerate the development of therapeutics and diagnostics. The Gator next-gen biolayer interferometry (BLI) instruments and biosensors enable real-time analysis of biomolecular interactions providing information on affinity, kinetics, concentration and epitope binning, etc. Most importantly, Gator Bio’s analytical capabilities enable better and faster characterization of drug candidates and viral vectors, thus providing greater value in drug development and gene therapy applications where existing methods have limitations in throughput, performance, and cost. The company is headquartered in Palo Alto, California with facilities in Shanghai and Suzhou, China. For more information, please visit www.gatorbio.com

Subodh Nimkar
Gator Bio
+1 650-575-0827
email us here
Visit us on social media:
LinkedIn

Gator Bio launches Gator^® Pro, a high throughput and high performance next-gen BLI system, for biotherapeutics discovery

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Gator Bio

January 13, 2023, 20:46 GMT

Gator Bio aims to set a new industry standard for high throughput and accurate kinetics, epitope binning and quantitation for biotherapeutics discovery

PALO ALTO, CA, UNITED STATES, January 13, 2023 /EINPresswire.com/ — Today, Gator Bio, Inc. announced the launch of Gator^® Pro, a high throughput and high performance BLI system for biotherapeutics development. The system is capable of 32-channel high frequency data acquisition in parallel. Combined with the new Gator^® Screener software, the total solution delivers high-quality kinetics and epitope binning results in fraction of the time taken by traditional BLI and SPR systems.

The new high throughput Gator^® Pro expands the current next-gen BLI instrument portfolio that includes the 8-channel Gator^® Prime and Gator^® Plus systems.

“Antibody and protein therapeutics developers repeatedly ask for more efficient BLI solutions that combine throughput and performance. We believe Gator^® Pro addresses those needs. It can also complement SPR as a high throughput screening tool.” said Dr. Hong Tan, CEO of Gator Bio. “We are singularly focused on delivering easy, efficient and effective solutions that would enable scientists to develop biotherapeutics faster”.

Detailed info for the Gator^® Pro along with Gator Bio’s complete portfolio of next-gen BLI products can be found here.

About Gator Bio, Inc.
Gator Bio is a life sciences company providing analytical solutions to accelerate the development of therapeutics and diagnostics. The Gator instruments and biosensors enable real-time analysis of biomolecular interactions providing information on affinity, kinetics, concentration, and epitope binning, etc. Most importantly, Gator Bio’s analytical capabilities enable better and faster characterization of drug candidates and viral vector analytics, thus providing greater value in drug development and gene therapy applications, where existing methods have limitations in throughput, performance, and cost. The company is headquartered in Palo Alto, California. For more information, please visit www.gatorbio.com.

SOURCE Gator Bio, Inc.
Media and Investor Relations Contact:
Subodh Nimkar, Marketing Director
[email protected]
+1 650 575 0827

Subodh Nimkar
Gator Bio
+1 650-575-0827
email us here
Visit us on social media:
LinkedIn
YouTube

The AAV Titer and Empty/Full Ratio Determination in Upstream Crude Samples is Challenging. The New Gator Assays Offer a Practical Cost-Effective Solution

SAN JOSE, CA, UNITED STATES, November 15, 2022 /EINPresswire.com/ —

Today, Gator Bio, Inc. announced the launch of Gator^® AAV Ratio Kit, High Sensitivity AAV Kit and High Sensitivity AAV9 Kit for empty vs full ratio determination and titer, respectively. The assays run on FDA 21 CFR Part 11 compliant Gator^® Prime and Gator^® Plus biolayer interferometry (BLI) instruments. This is the only automated single platform solution in the market capable of accurate and easy determination of titer and empty vs full ratios at every stage of the AAV development and production.

The automated Gator^® High Sensitivity AAV Kit and High Sensitivity AAV9 Kit enable titer determination with the widest dynamic range for serotypes 1-10 in crude and purified samples. This kit fits into every stage of the AAV processing workflow, and the titer measurements correlate well with the gold standard methods.

The Gator^® AAV Ratio Kit is an innovative empty vs full method that easily and accurately measures ratios from 5-100% full capsids in crude and purified samples. Along with its simplicity, good AUC correlation, short time-to-result, and cost effectiveness, the Gator^® AAV Ratio Kit will fundamentally change how this analysis is routinely done.

Enabling gene therapy developers and manufacturers with cutting-edge tools is Gator Bio’s focus. “There are many challenges and new demands in AAV and other vector analytics, and we are dedicated to developing practical solutions leveraging Gator’s next-gen BLI and novel chemistry know-how” said Dr. Hong Tan, CEO of Gator Bio. “We are singularly focused on delivering easy, efficient and cost-effective solutions”.

Detailed info for the newly released solutions along with Gator Bio’s complete portfolio of products can be found at –https://www.gatorbio.com/applications/gene-therapy/

About Gator Bio, Inc.
Gator Bio is a life sciences company providing analytical solutions to accelerate the development of therapeutics and diagnostics. The Gator instruments and biosensors enable real-time analysis of biomolecular interactions providing information on affinity, kinetics, concentration, and epitope binning, etc. Most importantly, Gator Bio’s analytical capabilities enable better and faster characterization of drug candidates and viral vector analytics, thus providing greater value in drug development and gene therapy applications, where existing methods have limitations in throughput, performance, and cost. The company is headquartered in Palo Alto, California. For more information, please visit www.gatorbio.com.

SOURCE Gator Bio, Inc.
Media and Investor Relations Contact:
Subodh Nimkar, Marketing Director
[email protected]
+1 650 575 0827

Subodh Nimkar
Gator Bio
email us here
+1 6505750827
Visit us on social media:
LinkedIn

Gator Bio is proud to announce achievement of ISO 9001:2015 certification for our manufacturing facility, a globally recognized certification of quality management system standards.

This further communicates Gator Bio’s commitment to develop processes ensuring consistency, reliability, and accountability across our business operations. By achieving this level of certification, Gator Bio continues to demonstrate our competence and desire to effectively and efficiently provide a quality management system while continuously improving our products, services, and internal processes. We strive to consistently meet and exceed expectations of our customers.

AAVs are common, harmless viruses that readily infect humans, therefore being very effective gene therapy vectors. Precise and accurate AAV titer quantitation is crucial to AAV based gene therapy, and as of now there is a lack of standardized quantification method across different production systems and laboratories.

What are AAVs and how are they used?

Adeno-associated viruses (AAVs) are a group of viruses that infect humans and primates. AAVs are very small (around 20 nm in diameter) and cannot replicate without the help of other viruses. They score low on a scale of virus complexity:  just consisting of a protein shell with a small single-stranded DNA payload. While AAVs produce a mild immune response in most humans, they’re not known to cause any disease. They also appear to be very common in human populations, with around 50% – 80% of human populations exhibiting seropositivity for antibodies directed against AAV capsid proteins.¹

These qualities make AAV serotypes (in particular AAV2) excellent  candidates for gene therapy, which is used to treat disease and genetic disorders by replacing ‘faulty’ copies of a gene with ‘healthy’ ones delivered by a viral vector. ²

What is AAV quantitation?

The use of AAVs in gene therapy applications has increased steadily over the last 20 years, and today AAVs are one of the most actively investigated gene therapy vehicles. Scientists have successfully used “recombinant” AAV (i.e., engineered versions of AAV that lack viral DNA) as essentially protein-based DNA delivery systems to treat a variety of conditions via gene therapy.

The development of safe and effective,  AAV gene therapies can only be accomplished if AAVs are accurately characterized and quantitated.⁵ The commercial demise of the first European-approved AAV-based gene therapy product due in part to cost issues highlighted the need for efficient AAV vector manufacturing and downstream processing.⁶ , which needs standardized methods to quantitate and characterize AAV vectors.

Challenges in AAV Quantitation

Multiple methods have been – and still are – employed to quantitate AAV capsid and genome titer. These include   enzyme-linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (qPCR), droplet digital PCR (ddPCR), and high-pressure liquid chromatography (HPLC).^7,8 These techniques are not only time-consuming; but  the lack of standardized methods for AAV quantification makes it difficult to compare yields from different production systems and across laboratories..⁹

AAV Quantitation from Gator Bio

Gator Bio has developed a unique solution to the challenge of accurate and efficient AAV quantitation.

Gator AAV probes are high-specificity antibody-based biosensors enabling the direct capture and rapid quantitation of different AAV serotypes in crude lysates, column eluates, and cell culture supernatants. Offering high accuracy and wide dynamic range (10⁹ – 10¹³ VP/mL for most AAV serotypes); Gator AAV probes provide a fast and efficient alternative to relatively time-consuming and labor-intensive traditional methods like ELISA. The reusability and crude sample tolerance of these probes make the method cost-effective and suitable for deployment at multiple stages in the development and manufacture of AAV-based gene therapies.

Gator Bio is a world-leading developer of BLI systems and solutions. To find out more about AAV quantitation solutions from Gator Bio, get in touch with us today.

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References and Further Reading

1. Grieger, J. C. & Samulski, R. J. Adeno-associated Virus as a Gene Therapy Vector: Vector Development, Production and Clinical Applications. in Gene Therapy and Gene Delivery Systems (eds. Schaffer, D. V. & Zhou, W.) vol. 99 119–145 (Springer-Verlag, 2005).
2. Zinn, E. & Vandenberghe, L. H. Adeno-associated virus: fit to serve. Curr Opin Virol 8, 90–97 (2014).
3. Naso, M. F., Tomkowicz, B., Perry, W. L. & Strohl, W. R. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs 31, 317–334 (2017).
4. How does gene therapy work?: MedlinePlus Genetics. https://medlineplus.gov/genetics/understanding/therapy/procedures/.
5. Dobnik, D. et al. Accurate Quantification and Characterization of Adeno-Associated Viral Vectors. Front. Microbiol. 10, 1570 (2019).
6. Ai, J., Ibraheim, R., Tai, P. W. L. & Gao, G. A Scalable and Accurate Method for Quantifying Vector Genomes of Recombinant Adeno-Associated Viruses in Crude Lysate. Human Gene Therapy Methods 28, 139–147 (2017).
7. D’Costa, S. et al. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR. Mol Ther Methods Clin Dev 5, 16019 (2016).
8. Dorange, F. & Le Bec, C. Analytical approaches to characterize AAV vector production & purification: Advances and challenges. Cell and Gene Therapy Insights 4, 119–129 (2018).
9. Aucoin, M. G., Perrier, M. & Kamen, A. A. Critical assessment of current adeno-associated viral vector production and quantification methods. Biotechnology Advances 26, 73–88 (2008).