Feliza Mirasol

American Society of Gene and Cell Therapy’s (ASGCT’s) 27th Annual Meeting

, Alex Meola, associate director of AAV Downstream Process Development at Oxford Biomedica, showed that hydrophobic interaction chromatography (HIC) is a useful tool for separating non-deamidated capsids from deamidated capsids, according to studies conducted by his team. The ASGCT annual meeting is occurring on May 7–11, 2024 in Baltimore, Md.

Meola emphasized how the focus on post-translational modification of capsid proteins of the adeno-associated virus (AAV) is growing in the process and analytical development space for gene therapies. The industry has been particularly focusing on how the deamidation of capsid protein from viral protein 1 (VP1)-specific residues, including the N57 domain, is linked to a loss of 

“Temperature, pH, and storage time have been identified as key factors causing this phenomenon,” Meola. “In our quest at Oxford Biomedica—to try and resolve this—we have a static binding capacity residence screening protocol that we run where we look at a variety of different residents. In this case, we decided to look at salt-tolerant residents,” he stated.

Besides process control to prevent deamidation, Meola’s team hypothesized (2) that extrusion of VP1 and deamidation of N57 are related phenomenon and result in generating empty, partial, and full capsids, which significantly influences the ability of the anion exchange chromatography (AEX) process. AEX’s ability to remove empty capsids and deliver functional AAV is thus compromised.

Meola noted that charge-driven separation techniques have not provided the resolution required to differentiate between deamidated and non-deamidated capsid species. Because of this, his team highlighted novel approaches that can be applied to the AEX process to address the complex challenge of removing both empty capsids and deamidated intact capsids. “Controlling strategies in the upstream and downstream processes should be studied and defined at the early stage of process development to limit onset of deamidation in order to minimize product related impurities and loss of vector functionality,” Meola stated.

One of the team’s first approaches was to use HIC to separate distinct peaks with near baseline resolution. After separation, each peak was isolated and individually reprocessed with AEX. “Surprisingly, we discovered that HIC resolved two different species of AAV capsids with near baseline resolution. Each species was reprocessed on AEX, and all product quality attributes were assessed for the intermediate peaks that were generated. We found that the capsids that were more hydrophobic were also more negatively charged,” he explained.

Furthermore, data from liquid chromatography–mass spectrometry analysis demonstrated that the more hydrophobic and more negatively charged capsids exhibited significant levels of VP1-specific N57 deamidation, which has been linked to a loss in gene expression. It was demonstrated, therefore, that the extrusion of VP1 exposes the hydrophobic phospholipase A2 domain on the VP1 unique region, a phenomenon that would make VP1 N57 residue exposed to solvent and thus susceptible to deamidation.

The team was thus able to demonstrate that HIC can be a useful method to separate non-deamidated capsids from deamidated capsids. Meola also stated that the team is working on a novel, patent-pending method to remove deamidated species while simultaneously enriching full capsids on the AEX process.

1. Giles, A.; Lock, M.; Chen, S. J.; et al. Significant Differences in Capsid Properties and Potency Between Adeno-Associated Virus Vectors Produced in Sf9 and HEK293 Cells. 

 (19–20),1003–1021. DOI: 10.1089/hum.2022.116
2. Meola, A.; Aguiar, A.; Chong, C.; et al. Not Created Equal: Impact of Deamidation on Capsid Heterogeneity and Anion Exchange Chromatography (AEX) Performance. Paper Abstract. annualmeeting.asgct.org (accessed May 8, 2024).

Articles

The separation of deamidated capsids from non-deamidated capsids can be achieved using HIC, according to studies done by a team at Oxford Biomedica.

Research Shows that HIC Can Effectively Separate Deaminated Capsids from Non-Deaminated Capsids (ASGCT 2024)

Bristol Myers Squibb and Cellares, a US-based integrated development and manufacturing organization (IDMO) specializing in cell therapy manufacturing, announced on April 22, 2024 that they have established a new agreement to reserve global capacity and supply for manufacturing chimeric antigen receptor T cell (CAR-T cell) therapies. The deal is valued at up to $380 million in upfront and milestone payments. 

Cellares Cell Shuttle is More Valuable Than the Sum of the Value of the Individual Parts

 with Fabian Gerlinghaus, CEO and co-founder, Cellares, on 

Under the agreement, Cellares will optimize, automate, and tech-transfer select CAR-T cell therapies from Bristol Myers Squibb onto Cell Shuttle, Cellares’ automated and high-throughput cell therapy manufacturing platform. Cellares will dedicate multiple Cell Shuttle and Cell Q systems, including fully automated, high-throughput quality control, to Bristol Myers Squibb for exclusive use. These Cell Shuttles and Cell Q systems will be deployed in Cellares’ smart factories located in the United States, European Union, and Japan.

In a company press release, Bristol Myers Squibb stated that, “Manufacturing cell therapies is both operationally and technically complex. Because cell therapies are rapidly transforming the way many different diseases are treated, the demand for these treatments is increasing significantly.” The collaboration with Cellares enables Bristol Myers Squibb to expand manufacturing capacity to meet the demand required from its diverse range of cell therapies. Cellares provides a platform technology that is scalable and carries the potential to improve turnaround times, according to Bristol Myers Squibb in its press release.

“The agreement with Cellares is our latest step forward in support of our comprehensive strategy to unlock the full potential of CAR-T therapy to deliver transformative treatments to as many patients as possible, as quickly as possible,” said Lynelle B. Hoch, president, Cell Therapy Organization, Bristol Myers Squibb, in the press release. “Our collaboration with Cellares strengthens our existing internal manufacturing capabilities for CAR-T cell therapies by giving us access to the first end-to-end fully automated cell therapy manufacturing platform, to help ensure we meet the high demand for these differentiated treatments, now and in the future.”

This agreement builds on existing collaborations between Bristol Myers Squibb and Cellares. Bristol Myers Squibb participated in Series C financing in August 2023 to fund the launch of Cellares’ commercial-scale 

 in Bridgewater, NJ, for cell therapy manufacturing, which, according to Cellares, is the first of its kind. The 118,000-ft

 smart factory is expected to be good manufacturing practice-ready in the second half of 2024. The facility is designed to integrate advanced robotics, purpose-built technology, and interconnected software and will be capable of producing 40,000 cell therapy batches per year (1). Also in August 2023, Bristol Myers Squibb joined Cellares’ Technology Adoption Partnership (TAP) Program to evaluate the Cell Shuttle’s automated manufacturing capabilities.

“This agreement with Bristol Myers Squibb is aligned with our strategy of establishing a global network of high-throughput, automated Smart Factories to meet the growing and worldwide demand for cell therapies,” said Fabian Gerlinghaus, CEO and co-founder of Cellares, in the company press release. “We look forward to demonstrating how our innovative technology’s emphasis on standardization will accelerate commercial-scale manufacturing and worldwide deployment. Our collaboration with Bristol Myers Squibb and our collective expertise furthers our mission to accelerate access to life-saving cell therapies for patients globally."

Gerlinghaus went into further detail about the development and analytical planning behind the company’s cell therapy manufacturing platform and the importance of automation to the manufacture of cell therapies in 

Bristol Myers Squibb Joins Cellares Program Focused on Automated Cell Therapy Manufacturing as Cellares is Launched as First IDMO

In a new agreement, Cellares will utilize its Cell Shuttle fully automated cell therapy manufacturing platform to manufacture select CAR-T cell therapies under development by Bristol Myers Squibb. 

Bristol Myers Squibb to Access Cellares' Cell Therapy Manufacturing Platform in New $380 Million CAR-T Cell Therapy Deal

Twist Bioscience, a synthetic biology and genomics company, announced on Nov. 14, 2023 that it has launched Express Genes, a new gene synthesis service with a quick turnaround time of five to seven business days. The service, housed in the company’s Wilsonville, Ore., manufacturing facility allows for the rapid production of genes.

Twist’s technology involves the miniaturization of the chemical reaction that creates DNA. The company does this using its silicon-based DNA synthesis platform coupled with expertise, software, honed processes, and an expanded layout at its Wilsonville facility. The technology allows the company to deliver clonal genes and gene fragments at scale and with rapid turnaround times.

“Twist Express Genes is our first new offering enabled by the increased capacity and streamlined processes in our Wilsonville facility. We are now able to deliver the same NGS [next-generation sequencing]-verified clonally perfect genes that our customers have come to expect from Twist in about half the time, at an unprecedented scale. We offer the rapid turnaround that our customers need in multiple formats and at the same speed for one or thousands of genes. With this new offering, we will focus initially on our current customers who we know are making some of their own genes,” said Emily M. Leproust, CEO and co-founder of Twist Bioscience, in a company press release. “We look forward to enabling the ‘Maker’s Market’ to continue to push the boundaries of what’s possible, working to uncover new therapeutic modalities for devastating diseases, ushering in sustainable applications for enzyme engineering and chemical development, and much more. They clone their own genes because otherwise they would have to delay their research while waiting for genes they ordered, and now they won’t have to make that compromise.”

“We surveyed several hundred gene users and found that in addition to those who make their own genes entirely, a large portion of respondents both purchase DNA from a supplier and in parallel make their own,” said Anne St. Louis, director at Percepta Associates, in the press release. “These data suggest that there is a market to capture from researchers in industry and academia who currently use vendors for only part of their total gene production.”

"As a gene therapy startup developing programmable RNA medicines, we routinely generate diverse and customized DNA sequences to advance our technologies and enable application to multiple disease targets. Being able to order sequence-verified large synthetic gene constructs in just one week expands what we can screen and allows faster iterations of the design-build-test cycle," said Susan Byrne, senior principal scientist, Shape Therapeutics, in the press release.

Twist Bioscience now offers its Express Genes rapid gene synthesis service at its Wilsonville, Ore., manufacturing facility.

Twist Bioscience Launches Gene Synthesis Service

FUJIFILM Diosynth Biotechnologies has completed its new facility in Darlington, United Kingdom, which is now operational and can carry out current good manufacturing practice (CGMP) manufacturing of viral vector-based gene therapies, oncolytic viruses, and viral vaccines for clinical trials. The completion of this facility is part of the company’s investment strategy to address the increased demand for production of early stage clinical trial (first-in-human) investigational viral vector products, the company stated in a Sept. 14, 2023 press release.

The new CGMP site houses dedicated high containment clean rooms for viral vector manufacturing and segregated viral and non-viral suites. The facility holds a cell culture capacity of up to 200 L. The addition of the Darlington facility adds to the company’s global locations, which include College Station, Texas, and Watertown, Mass., in the United States (1).

“We are excited to continue working with our biopharmaceutical partners, including early-stage companies, to help them bring new gene therapies to market, which hold tremendous promise in revolutionizing treatment options for patients,” said Jonathan Haigh, head of UK site at FUJIFILM Diosynth Biotechnologies, in the press release. “Our advanced therapy integrated network, including an ongoing commercial viral gene therapy expansion in nearby Billingham, UK, allows customers to benefit from our end-to-end cell and gene therapy services provided by our highly skilled workforce.”

“In addition to providing increased manufacturing capacity for our early stage customers, we look forward to helping them efficiently navigate preclinical and Phase I hurdles by offering complementary services for fast, yet flexible drug candidate screening as well as feasibility studies,” Haigh added in the release. “This additional support will help biopharmaceutical customers create a simple, modular path to cGMP manufacturing, minimizing risk and streamlining drug substance and drug product manufacture for the clinic.”

1. FUJIFILM Diosynth Biotechnologies, Globally Integrated CGMP Production. 

FUJIFILM Diosynth Biotechnologies has finished its new viral vector gene therapy manufacturing facility in Darlington, United Kingdom.

FUJIFILM Diosynth Biotechnologies Completes New Viral Vector Gene Therapy Manufacturing Facility in UK

CN Bio, a organ-on-a-chip (OOC) company specializing in the design and manufacture of single-and multi-organ microphysiological systems (MPS), has partnered with LifeNet Health LifeSciences (LifeNet Health), a non-profit provider of all-human research solutions, to supply validated primary human cells. The cells will be used with CN Bio’s PhysioMimix OOC range of MPS.

Under the agreement, LifeNet Health’s MPS-validated, hepatic cells will be made available either within CN Bio’s non-alcoholic steatohepatitis (NASH) “in-a-box” kit or directly from LifeNet Health. The companies expect that this partnership will provide a streamlined solution for the recreation of CN Bio’s advanced 

 liver models and that the combination will maximize assay success.

According to a Sept. 6, 2023 company press release, CN Bio will use a stringent process to pre-qualify select hepatocyte, stellate, and Kupffer cells that are proven to thrive in extended 3D MPS culture. These cells will have to show that they maintain function and phenotype for a minimum of two weeks. It is known that lot validation in standard static 2D or 3D cell cultures does not guarantee the performance of primary cells in perfused MPS cultures. CN Bio carried out an analysis over a five-year period where it demonstrated that greater than 60% of hepatocyte cell lots across various suppliers failed to meet acceptable performance criteria for MPS assays. Thus, pre-validating cell lots for use in MPS assays is necessary, and it ensures that robust and reliable human-relevant data can be generated, according to the company press release. However, the validation process itself requires that additional resource, time, and budget be factored in.

“LifeNet Health and CN Bio share a commitment to helping researchers bring safer, more effective therapies to patients sooner, with no unnecessary animal testing,” said Tomasz Kostrzewski, chief scientific officer, CN Bio, in the press release. “In our studies, LifeNet Health’s cells have consistently shown high success rates, and we are confident this will lead to more reliable, reproducible results. Our partnership represents an important milestone for existing customers, alongside those looking to adopt OOC technology into their own labs. Validation of cells is a costly and laborious process, but one that is imperative for assay success. Harnessing our combined technical expertise and customer support, we can offer researchers a more complete solution to easily recreate our industry-leading liver models, removing the risk of failed experiments due to untested cells and the burden of in-house quality control testing—so that they can focus their efforts on new discoveries.”

“This partnership represents a joint investment to set a new standard for cell quality across the industry, enabling customers across a broad range of market segments to realize the potential of OOC, without the need for costly in-house validation,” said Ed LeCluyse, chief scientist, LifeNet Health LifeSciences, in the release. “Our cells are fully characterized for quality and performance, to ensure they can perform within advanced platforms, to more effectively mimic 

CN Bio and LifeNet Health LifeSciences have teamed up to provide direct access to a portfolio of highly characterized hepatic cells for the creation of advanced liver-on-a-chip assays.

CN Bio and LifeNet Health LifeSciences Partner to Supply Pre-Validated Cells

On Aug. 1, 2023, US-based iTolerance, an early-stage regenerative medicine company, and Lithuania-based Northway Biotech, a contract development and manufacturing organization, announced that they have entered into an agreement for the manufacturing of Streptavidin-FasL (SA-FasL) fusion protein, an important component of iTolerance’s proprietary iTOL-100 platform technology.

According to a company press release, the SA-FasL fusion protein has demonstrated promising potential in pre-clinical studies; it was shown to establish durable, localized immune tolerance, which would thus enable implanted tissue or organoid, or a cell therapy, to function as a viable replacement for damaged native cells.

Under the agreement, Northway Biotech will use its expertise to develop a robust manufacturing process for SA-FasL. Process development work will involve the production of a master cell bank, the development and qualification of appropriate analytical methods, the optimization of formulation, and the development and scaling up of the production process. Northway Biotech will also be responsible for manufacturing a current good manufacturing practice-produced batch of drug substance for clinical studies.

“We believe this strategic partnership is not only a significant step for iTolerance, but also in developing advanced regenerative medicine technologies. We are incredibly delighted to establish this important partnership with Northway Biotech … to manufacture the clinical supplies necessary for our first in human clinical study for our lead program, iTOL-102,” said Anthony Japour, CEO of iTolerance, in the press release. “We look forward to working closely with the Northway Biotech team to advance our iTOL-100 technology and progress towards providing treatment options that have the potential to cure diseases without the need for chronic immunosuppression.”

“We believe this partnership is an exciting development in the regenerative medicine field, and we are honored to play a role in advancing this important work. With a commitment to meeting contractual timelines, our primary objective is to ensure the punctual delivery of these crucial materials to our esteemed partners at iTolerance,” said Vladas Algirdas Bumelis, CEO and chairman of the board at Northway Biotech, in the release.

Under a manufacturing agreement, Northway Biotech will develop a manufacturing process for iTolerance’s fusion protein in development for regenerative therapy.