Biotech Week Boston, held at the Hynes Convention Center in Boston, MA, was a fantastic amalgamation of scientific conferences, training courses, and keynotes all focused on cell bioprocessing. The theme of this year’s Cell and Gene Therapy Bioprocessing and Commercialization conference was “transforming innovative research to drive the future of cell & gene therapy by relieving bottlenecks and pain points in clinical development, manufacture and commercialization of cell, gene and immunotherapies”. With so many changes in cell and gene therapy field stemming from the commercialization of groundbreaking new treatments (CAR-T therapies), healthcare professionals, researchers and therapy manufacturers alike are trying to balance safety, cost and treatment logistics to bring these technologies to patients as fast as possible. There were some of the major themes repeated throughout the conference that show the bioprocessing community is working to achieve these goals:

• process intensification (viral vectors)
• incorporation of automation and single-use technology
• leveraging technologies from other industries (i.e. antibody production technology and stem cell transplantation centers)

Inspiring keynote speakers, such as Bob Langer (David H. Koch Institute Professor at MIT) and George Yancopoulos (President & Chief Scientific Officer, Regeneron Pharmaceuticals) rounded out a diverse and informative conference.

The conference kicked off with a great panel discussion hosted by the Cell Culture Dish for the launch of the eBook titled “Key Considerations in Gene Therapy Manufacturing for Commercialization”. A group of industry experts were gathered to field questions regarding the current state of the art with viral vector manufacturing and discuss where the field is headed. Audience members asked a variety of questions ranging from the cost of cell therapies to whether viral vectors would continue to be prevalent for manufacturing these therapeutics. To download the eBook, please visit: https://cellculturedish.com/genetherapybook/

A Platform Production Process for Manufacturing Viral Vector and Vaccine Therapeutics using Vero Cells

One of the biggest struggles for cell and gene therapy manufacturing is meeting the demand for viral vectors. Manufacturers are investigating methods to be able to meet the needs of cell therapy makers. John Madsen, PhD, Head of Process Development Operations at FUJIFILM Diosynth Biotechnologies, gave a talk on a new Vero cell line platform under development for viral vectors production with the potential to significantly increase productivity for a variety of viral vector types compatible with single-use technology. The Vero cell line was established from kidney epithelial cells isolated from the African green monkey and is already in use for the manufacture of five FDA-approved vaccines. It is well suited for viral vector production because of the following attributes:

• Permissive to infection by many types of viruses
• Safety tested in humans
• Can be cultured in chemically defined medium
• Cells are amenable to high density culture
• Cells can be suspension-adapted or adherent (maintained on microcarriers)

An important point of discussion was the idea of process intensification. The concept is to increase the specific activity of the producer cell, whether through improvements to culture media to increase cell viability or using more advanced techniques like gene editing to turn off the “death” switch in the cell to allowing them to grow at higher than normal densities. Process intensification allows a manufacturer more flexibility and time in process development before scale up is required to meet vector demands. If the cells are highly productive, the production scale can be kept small before needing to invest in equipment, etc to scale up to larger volumes. This saves money and time for therapies in the clinical pipeline with very compressed timelines.

The Balancing Act: Engineering vs. Tinkering Platform Processes with GMP Manufacturing in Mind

Another approach to improving viral vector yields was presented by Pratik Jaluria, PhD, Executive Director, Process Development and Manufacturing at Adverum. Specifically addressing adeno-associated virus (AAV)-based vector systems, his team developed an adaptable baculovirus/Sf9 insect cell expression platform to work across multiple therapeutic constructs, at varying scale. Two separate, infectious baculovirus stocks are made; one with the rep/cap gene to make viral capsid and replicate the genome while the second has the desired gene product (i.e. gene of interest), which are them used to infect naive Sf9 cells. Sf9 host cells replicate vector DNA much more efficiently than HEK 293 cells providing the ability to generate high titer at a smaller scale more rapidly provides a high degree of flexibility in their process.

Through the talk, I was struck by the forethought of decisions made during the platform process development early on that would facilitate future scale up needs of the system. Incorporating technologies and upstream/downstream changes early during process development makes it significantly easier to rapidly increase the scale of a platform. Any alterations in manufacturing protocols later in a cell therapy’s development would require validation and comparison studies that could drastically slow down timelines.

For example, Pratik explained that they opted to use ion exchange chromatography (IEC) during process development instead of the typical ultracentrifugation polish step because IEC is a much more scalable technology. Incorporating this change is much easier now is much easier than in later clinical stages when the manufacturing process becomes more fixed.

Future work by the group to optimize the platform include:

• Scale up to 200L
• Addition of extra IEC post-polish to improve robustness (improve removal of empty capsids, etc)
• Elimination of ultrafiltration/diafiltration step with changes in buffer alone to minimize handling steps
• Incorporation of in-line technologies such as OT2 platform (automated liquid handling to facilitate DNA extraction for qPCR analysis to determine AAV titer), TECAN and ambr systems
• Leveraging monoclonal antibody manufacturing knowledge and developing specific analytical tools for in-process testing

From both John and Partik’s talks, I got the sense that viral vector manufacturers are heavily invested in manufacturing smarter and building their process knowledge to allow better control of manufacturing steps. Rather than just simply scaling up to meet vector demands, they are looking at “process intensification methods” that allow for the manufacture of viral vectors at high titers in smaller volumes, at a much more rapid pace than ever before.

Industrialization of Viral Vector Manufacturing for Gene Therapies: Challenges and Progress

Rachel Legmann, PhD (Manager of Cell Culture Process Development Lab and Application Specialist) from Pall Corporation presented a great case study for viral vector optimization for a cell and gene therapy client as part of their CRO services utilizing their iCELLis and Xpansion platforms. This client was in preclinical stages for Type 1 diabetes treatment using adenoviral vector-based methods to modify liver cells. It was an informative talk for any company looking to contract out their process development to experienced manufacturers who can take research level flat plasticware cultures to bioreactor production.

Flask → iCELLis Nano → iCELLis 500 → Xpansion bioreactor

Leveraging knowledge of a CRO can be beneficial to facilitate optimization and scale up of a new therapy reducing the burden on the therapy owners.

GPS for Cell Therapy: Optimizing Treatment Efficiency and Safety via Enforced Cell Navigation

Switching gears to CAR-T therapies, I attended a Biotechne-sponsored talk by Robert Sackstein, PhD (Professor, Departments of Medicine and Dermatology, Harvard Medical School; Director, Harvard Program of Excellence in Glycoscience; and Co-Director, Harvard Glycoscience Center) on the topic of therapeutic cell migration to target tissues within the body. With CAR-T therapies, there are often patient complications from treatment related to therapeutic cell dose which ranges from 25 – 250 x 10⁶ cells/L blood volume (CRS in >85% recipients; CRES in ~50% recipients).

The goal of their research was to improve delivery of vascularly administered culture-expanded CAR-T cells to sites where they are needed. Homing is an active process of ‘extravasation’ dictated by expression of specific receptors of the cell in the blood flow that engages pertinent counter-receptors on target tissue endothelial cells. Regulatory steps in homing:

Step 1: The “Molecular Brake” where cells in the blood flow are recruited to the endothelium by E-selectin (CD62E); results in cell “rolling” on surface of endothelial cells

Step 2: Integrin Activation by chemokines (low affinity binding)

Step 3: Stable Adhesion to endothelial cells (high affinity binding)

Step 4: Migration through endothelium into target tissues

In adults, only hematopoietic stem cells (HSCs) express E-selectin ligands allowing them to home to the bone marrow microenvironment. These ligands are absent from culture-expanded T and CAR-T cells. So, how can we modify CAR-T cells so that they will home to target tissues rather than non-specifically localizing to any tissue after administration?

Localization (random entrapment) ≠ Homing (specific recruitment to site)

The most potent receptor for E-selectin on human cells is Hematopoietic Cell E– and L-selectin Ligand (HCELL), and receptor binding occurs at the glycan site sLeX (Sialyl Lewis X or CD15s) on the ligand.

In order to convert endogenous CD44 on lymphocytes to HCELL, the α (2,3) Sialyl lactosamine group is converted to sialyl Lewis X (CD15s) using fucosyltransferase enzymes (Biotechne/R&D Systems). This exofucosylation allows for circulating lymphocytes to bind endothelial E-selectin allowing for tissue specific homing.

The fucosyltransferase is an example of a GPS (Glycosyltransferase-Programmed Stereosubstitution) reagent available from Biotechne/R&D Systems to create stereospecific glycan modification(s) while maintaining cell viability and function; something that is not possible when these alterations are made using synthetic organic chemistry.

What’s the clinical relevance for this improved tissue delivery system for CAR-T cells?

• Decrease cost of cell expansion (substantially fewer cells for treatment are needed for therapeutic effect)
• Decrease toxicities related to cell dose (cells will home specifically to target tissues reducing off-target effects)
• Make cell-based therapies more efficient, affordable and safer

CAR-T therapies are the subject of popular media particularly with the approval of Novartis’ Kymriah™ and Kite/Gilead’s Yescarta® in 2017. Undoubtedly, improving the safety and efficacy of these therapies while reducing cost is a win for patients and therapy makers alike.

Strategies for Creating Standardization Across the Cell and Gene Therapy Industry

Jamie Margolis, PhD (Director, Product Development Operations at Be The Match BioTherapies) gave a talk on supply chain logistics for complex product delivery processes and the need for standardization. The company has a history of managing stem cell transplants from organizing collection of donor materials to transportation of cells (live or cryopreserved) to health care providers at treatment centers. There are many similarities in the logistics of this process that can be leveraged for cell and gene therapy products. This complex system is made even more so with quality and/or schedule disruptions that impact the entire supply network due to the high degree of interconnectivity between all contact points.

• Quality of start material from donors is highly variable – center to center variations in apheresis protocols
• Logistics risks – patient health for apheresis, transportations disruptions (i.e. weather), scheduling conflicts between collection centers and therapy manufacturers
• Capacity – do you have an apheresis sample when a batch needs to be manufactured?
• Regulatory requirements – multiple audits by various clinical trial companies at the same centers disrupts collection schedules

The company has a standardized technology platform with an existing network of verified donors worldwide and the ability to manage the logistics of moving samples to and from various points in the supply chain such that problems due to ensure schedule disruptions can be mitigated. Additionally, they have licensed audits available to companies for many of the major apheresis centers to reduce the need for multiple, independent audits.

Stem cell transplantations have been ongoing for decades and it is promising to see how current technologies can be applied to the new cell and gene therapy treatment paradigm. Particularly with the need for short turn-around times from patient apheresis to reinfusion of the cell product, real-time supply chain management for all stakeholders is a must.

Final Remarks

Overall, I think the concept of PAT has really taken hold and manufacturers are looking at all aspects of their manufacturing processes to see which steps can be optimized, eliminated or changed to facilitate the needs of the field as demand continues to increase. Also, the emphasis on improving efficiency (process intensification), flexibility (in scale), safety and dealing with the logistics burden of these complex therapies was prevalent in many of the talks. By no means is this an exhaustive summary but some of the main themes highlighted throughout the conference as a whole. Other ‘take home’ points from talks I attended include:

• Tighter control of raw materials (switch to defined, serum-free culture media) and process (build process knowledge) because the quality of start cells is highly variable
• Digital technology integration for in-line testing/paperless records (i.e. Roche’s Smartline Data Cockpit)
• Low cost automation is a priority
• Companies can drive value with process and facility intensification
  • Manufacturing facility designs are moving to open architecture format (vs. many walls and closed rooms) to stay flexible while optimizing cost and capital
  • Next generation single-use manufacturing platforms allow for a smaller footprint with similar output potential as traditional stainless steel
• Upstream process intensification focus:
  • Increased titer and productivity
  • Shorter cycles
  • Media optimization
  • Dynamic control of perfusion rates
• Downstream process intensification focus:
  • Multi-column chromatography (protein A)
  • Automated VI
  • Pool-less integrated processing (CEX/AEX)

Until next year!

Don’t miss the Cell Therapy Manufacturing and Gene Therapy Congress, December 4-6, 2018, Amsterdam