NCBiotech's BPD Group Tackles Cell and Gene Therapy Challenges

Research and development of cell and gene therapies is happening at a breathtaking pace at dozens of U.S. companies, many based in North Carolina.

As early as next year, the FDA expects 200 Investigational New Drug applications (INDs) – in addition to the 800 current ones – from this niche of the life science sector alone.

However, to bring these therapies to patients, the companies that develop and manufacture them face ongoing challenges in key areas. The entire ecosystem is grappling with solutions related to product characterization, new safety risks, scalability, an FDA guidance system and manufacturing processes originally designed for large-molecule drugs and the large clinical trials associated with them. There is a pivotal need in cell and gene therapies for reliable, cost-effective manufacturing processes.

The North Carolina Biotechnology Center’s Biomanufacturing and Process Development (BPD) Exchange Group of industry professionals working in this space convened to address these topics for a symposium and vendor show earlier in April.

Six speakers from companies spanning work from upstream through the downstream of cell and gene therapies shared insights about how their organizations are pushing forward this exciting frontier of medicine. Ronna Dornsife, an instructional technology specialist at North Carolina Central University’s Biomanufacturing Research Institute and Technology Enterprise (BRITE) and member of the BPD Group’s steering committee, led the event attended by 164 like-minded professionals.

Fifteen vendors showed equipment, supplies and the myriad ways they support these specialized manufacturing processes. Far less standardized equipment and methods for integrated automation is in use for cell and gene therapy product manufacturing, compared with manufacturing of large-molecule biopharmaceuticals. Some of the speakers addressed this challenge.

Scaling Up

For example, critical bottlenecks are impeding growth of regenerative medicine, explained Katrina Adlerz, Ph.D., development scientist at RoosterBio, Inc. One is the availability of cells produced according to the FDA’s regulations for Current Good Manufacturing Practices (cGMP-compliant) for clinical development and translation.

There are over 900 registered clinical trials using mesenchymal stem/stromal cells (hMSCs, or human bone marrow stem cells) and hMSC-derived materials for therapeutic applications, each possibly requiring billions of cells, said Adlerz. Thus, there is an urgent need for development of economical biomanufacturing processes capable of generating these lot sizes.

RoosterBio is working on manufacturing solutions to enable this rapid clinical translation of hMSCs and hMSC-based therapies. Their goal is to simplify the technology by providing standardized stem cell product platforms.

Adlerz described the bioreactor (large vessel in which to grow cells at scale beyond the traditional dishes, test tubes or flasks) process developed by RoosterBio to the 50-liter lot size using high-volume XF (xeno-free) hMSC cell bands, an optimized XF fed-batch bioprocess media system and XF microcarriers. Xeno-free means containing no materials different from the species of the stem cells in the bioreactor. Scalable bioreactor culture systems under development provide the lot sizes required for clinical trials using hMSC and hMSC-derived materials and commercial therapies. They also offer significant time and cost savings compared with standard systems.

“We are continuing to think about how to scale up even further as the industry needs larger lot sizes,” said Adlerz.

She described the various adjustments necessary as they scaled up from one lot size to the next. For example, they moved to different systems for harvesting and separating cultured cells with the larger volumes and at the manufacturing stages of volume reduction and wash. They also engineered feed of culture media to overcome technical challenges and costs associated with the larger lot sizes.

“What we have seen with our customers is that this system allows them to cut down the time to clinic by half,” said Adlerz.

Ensuring Safety & Quality

Potential contamination at several points along the biomanufacturing process is of high concern in this work. Jeremy Smith, head of quality control at Promethera Biosciences, LLC, presented his company’s methods for reducing bioburden (unwanted bacteria) as it isolates cells recovered from healthy non-transplantable livers for therapeutics purposes. The livers arriving at Promethera’s GMP-compliant facility are not sterile, so the company must take measures to reduce risk of contamination entering its Class A clean room, as well as its final liver cell products. This even means that certain steps in the process must take place off-site.

Promethera develops and manufactures liver cell-based therapies to reduce the need for liver transplantation. It currently has a patented allogenic (donor) liver cell platform, HepaStem®, in a Phase IIa clinical trial to treat patients with Acute-on-Chronic Liver Failure (ACLF), which has potential to target other severe and more prevalent chronic liver diseases such as Non-Alcoholic Steatohepatis (NASH).  

Careful screening for eligibility occurs before a donor liver ever enters the Promethera facility. Transport media include three different sterile organ preservations solutions and packaging in three sterile bags for shipping on wet ice. There is subsequent testing for microbiologics, washing and soaking with buffers containing antibiotics to reduce contaminants. Upon arrival, a sample of the transport media are sent to a contract laboratory in Europe for testing for microbial contamination, including 14 days of culture. If a mold or yeast is identified, the liver is rejected. The final product developed using cells from the liver is also tested in the same way before release.    

Standardizing Platforms

CAR T (chimeric antigen receptor) therapy is one of the most exciting frontiers in cancer immunotherapy. It involves removing a patient’s own T immune cells, genetically modifying and then reinfusing them back into the patient to mobilize one’s own immune system to attack malignant cells.

Using a patient’s own cells (in the autologous method) – while being preferable for avoiding the potential for graft-versus-host disease – presents several challenges, however. Patients may not have enough of their own healthy T cells available for the process due to disease progression. Also, there is a delay in treatment for the genetic modifying process between removal of the patient’s T cells to reinfusion of the CAR T cells. 

Therefore, the industry seeks a safe “off-the-shelf” platform for genetically modifying healthy donor T cells (in the allogenic method) that can be manufactured, frozen and kept ready for injection as soon as a patient needs them. Mark Johnson, Ph.D., team leader for cell therapy at Precision Biosciences, presented about the company’s ARCUS® platform, which endeavors to do just that. ARCUS® is derived from a natural genome editing enzyme, called a homing endonuclease, which can be used to insert, delete or edit DNA.

Late last year, Precision Biosciences received FDA authorization to begin a study of its first clinical-stage product candidate, a cancer immunotherapy. The company has an allogeneic anti-CD19 CAR T therapy for two types of blood cancer: B-cell acute lymphoblastic leukemia (B-ALL) and non-Hodgkin lymphoma (NHL). The CD19 is a protein on the surface of immune B-cells encoded by the CD19 gene. It plays critical roles in immune B-cell signaling and is a biomarker and therapeutic target for leukemia immunotherapies. According to the Mayo Clinic, research is underway for the use of CAR T therapy in multiple myeloma as well.

On April 1, Precision closed a $145 million initial public offering (IPO) of stock and is currently in its legally mandated several-week “quiet period” following the IPO, so could not comment for this article.   

According to the Precision website, “Homing endonucleases are nature’s genome editing system. They are site-specific DNA-cutting enzymes encoded in the genomes of many organisms. These non-destructive enzymes trigger gene conversion events that modify the genome in a very precise way, most frequently by the insertion of a new DNA sequence. The ability to target a single DNA break in a complex genome and to achieve gene modification without random off-targeting makes homing endonucleases the ideal starting material for a therapeutic-grade genome editing technology.

“The backbone of the ARCUS® technology is the ARC nuclease – a fully synthetic enzyme similar to a homing endonuclease. It can be customized to recognize a DNA sequence within any target gene and optimized to control potency. ARCUS® can be used to insert, delete or edit DNA.”

Following FDA Regulations

Designing, implementing and follow-up of clinical trials involving cell and gene therapies is increasingly complex. For example, FDA requires 15 years’ follow-up of patients who participate. Stephanie Pierce, Ph.D., RAC, regulatory affairs scientist in the Office of Regulatory Affairs and Quality, Duke University School of Medicine, spoke about the regulatory obstacles for starting such trials and presented highlights of FDA’s newest guidance.

She opened, however, with a sober reminder about gene therapy’s “troubling past.” Alarming and tragic events in the late 1990s and early 2000s (an adult patient’s death from a massive immune reaction and halt of a pediatric trial due to adverse side effects) led to a recession of gene therapy. Nevertheless, research and development of gene therapy continued to move forward, but at a slower, more cautious pace, said Pierce. To cope with the surge in INDs and over 120 actively recruiting clinical trials in the U.S. for CAR T cell therapies alone, she said FDA is hiring more reviewers and releasing clinical guidances related to cell and gene therapy product development.   

For example, FDA guidance issued in 2018 noted that modifications to a vector (a biologic used as the vehicle to transport modified genes) may alter the risk profile of products that are currently considered lower risk. This matters in terms of the protocols investigators must design for the long-term follow-up required for cell and gene therapy clinical trial participants. Certain vectors require 15 years’ follow-up, others up to five years’ follow-up, with different levels of patient contact and testing over the duration of follow-up.

Pierce touched on other recent FDA guidances and the issues related to fitting cell and gene therapy products into a regulatory landscape designed for drug products. Many cell and gene therapy trials have relatively low numbers of subjects in comparison with the high numbers in traditional pharmaceutical trials. This means fewer manufacturing runs, which makes it difficult to determine the critical process parameters to ensure the required Critical Quality Attributes. Also, because the full scope of some of the diseases is not well understood, or the therapies are not expected to be cures, per se, she said FDA encourages development of novel endpoints. Pierce closed with 2019 highlights of FDA criteria for some expedited programs of review of regenerative medicine therapies.

Using Viral Vectors in Gene Therapy

Recombinant adeno-associated virus (AAV) is a virus that is not harmful and one of the most commonly used vectors for gene therapy. This method exploits the natural process of how viruses attack cells and introduce their own genetic material with instructions to replicate identical cells. Advantages of AAV include its high efficiency in infecting target cells, stable gene expression and lack of association with a strong inflammatory response. In 2017, the FDA approved the first ever U.S.-based gene therapy using AAV, Luxturna, to treat patients with a rare form of inherited vision loss. 

William (Billy) Kish, Ph.D., senior scientist in gene therapy bioprocess development at Bamboo Therapeutics, delivered a tutorial on adeno-associated virus (AAV) biology and both upstream and downstream bioprocessing methods. In 2016, pharmaceutical giant Pfizer of New York acquired Bamboo, a spinout of Asklepios Biopharmaceutical (“AskBio” for short), which is a gene-delivery technology company co-founded in 2003 by entrepreneurs Sheila Mikhail and R. Jude Samulski, Ph.D., director of the Gene Therapy Center at the University of North Carolina at Chapel Hill. Samulski was the first to clone AAV for therapeutic purposes. Bamboo is one of several companies spun out of AskBio, which has grown with the help of more than $700,000 in grants and loans from NCBiotech to support its research and commercial development.

According to Kish, different variations, or serotypes, of AAV are under development to target hemophilia, congenital heart failure, muscular dystrophies, cystic fibrosis, Parkinson’s, Alzheimer’s epilepsy, ALS and macular degeneration among other diseases. He covered the upstream processing topics of cell lines, transfection methods (artificially introducing nucleic acids – DNA or RNA – into cells) and bioreactor operations (the device or system in which to culture cells). He followed with downstream processing, including cell lysis (disruption of cell membranes), clarification (separation of molecules), density gradient ultracentrifugation (high-speed spinning to cause separation) and chromatography.

Different challenges present themselves along the entire process. For example, maximum release of AAV during the process requires that the membrane surrounding the cell nucleus must be lysed, or destroyed, by a low pH detergent. This releases the components of the cell nucleus, yet also introduces impurities. To separate, or clarify, the various molecules based upon differences in density, they use density gradient ultracentrifugation. However, this method presents concerns over scalability, operator safety, cleaning and calibration of the equipment.

Kish said that most downstream processes are currently dependent upon the different serotypes. Development of scalable platform-able unit operations is of high interest to the gene therapy industry. Because of the proprietary nature of the work underway, he could not provide details, but explained that Pfizer’s vision of gene therapy includes development of an AAV platform, cell lines, sophisticated vector analytics and scalable manufacturing.

This provided the perfect segue to the final presentation by Jacob Smith, director of process development at Asklepios BioPharmaceutical, Inc. (AskBio), so-named due to its schematic structure, which the company is pursuing for a safer, cheaper and more efficient alternative to plasmid DNA.

Plasmids are small, circular DNA sequences within a cell – found widely in bacteria – that do not contain chromosomes but are capable of replicating. They are used as base material for manufacturing vectors to carry edited genes in gene therapy. There is a limit, however, on the vendor capability to produce large enough quantities of bacterially derived plasmids to keep up with the pace of this growing area of medicine. Thus, there is an unmet need for a synthetic version. DoggyBone was developed by Touchlight Genetics Ltd., a UK firm with which AskBio recently has entered into partnership.

“Something AskBio did was invest early on in manufacturing. It was a good investment, because at some point everyone realized that you have to be able to make sufficient quantities to be able to treat a very rapidly growing industry,” said Smith. “Furthermore, it’s important to start early on with a scalable process with an established purification platform.” Smith focused a large portion of his talk on the Doggybone™ platform.

According to Smith, the Doggybone™ is “going to result in a paradigm shift in the raw materials used in the transient transfection process. It could replace the plasmid as the production starting material.”

One benefit this technology has is that none of the original bacterial material winds up in the final product. Other advantages they have observed are a yield increase and a timeline to make this material in weeks rather than months for plasmids. AskBio and Touchlight have a joint venture manufacturing facility in Spain, where they anticipate using the Doggybone to manufacture AAV.

The morning of the symposium, AskBio also announced receipt of $235 million in new funding to help advance and expand clinical trials, enhance its manufacturing capabilities and capacity and drive long-term growth, according to a company news release. TPG Capital and Vida Ventures are investing $225 million for a minority stake in the company, and AskBio’s founders and board members are co-investing $10 million.