Monthly Archives: July 2019

Mustang Bio’s gene therapy has tremendous potential to cure XSCID or Bubble Boy Disease based on study results from St. Jude



In this podcast, we interviewed Dr. Manny Litchman, President and CEO, Mustang Bio and Dr. Knut Niss, Chief Technology Officer, Mustang Bio about the exciting study results for their gene therapy candidate to treat X-SCID, why this disease is a good fit for gene therapy and next steps.

Show Notes

To begin the podcast, I asked Dr. Litchman about the study results in treating X-SCID or “bubble boy disease”, including current standard of care and why gene therapy is a good fit for this disease. He explained that X-SCID is an X linked rare genetic disease with mutation in gene responsible for insufficient immune system generation at birth. As a result, severe, recurrent and opportunistic infections occur and usually results in death by age one if left untreated. Current standard of care is Immune reconstitution by allogeneic stem cell transplant, however the transplant must be from a matched donor and the best donors are siblings. There are only about 15% matched sibling donor transplants at this time. Gene therapy would eliminate the need for a matched donor and would improve survival and quality of life for the patient.

Next I asked Dr. Niss about previous attempts at treating this disease with gene therapy and how their therapy is different. He described that in the early days of gene therapy gamma retro virus was sometimes used as the vehicle for delivery.  For the current gene therapy a lentiviral construct is used, which has safety features that the gamma retrovirus didn’t provide. In the previous attempt to use gene therapy for X-SCID, the patients were cured of X-SCID, but developed leukemia due to the use of the gamma retrovirus. Today gene therapies build many safety features into the construct, many of which are incorporated into the vector used in the current study.

I then asked Dr. Litchman to elaborate on the St. Jude study and the very positive results. He explained that the results were published in the New England Journal of Medicine, “Lentiviral Gene Therapy Combined with Low-Dose Busulfan in Infants with SCID-X1,” by St. Jude. The study was a single arm study in newborn patients who were diagnosed with X-SCID. The results were striking as all patients had multi-lineage immune reconstitution. In addition, all patients that had infections prior to gene therapy cleared completely, with levels of IgM that are produced in B cells normalized in 7 patients. Four patients were able to stop IV immunoglobulin (pooled antibodies that these patients receive to prevent infections) and 3 patients had a normal response to childhood vaccinations. The safety profile was excellent with no leukemia and no transfusions required.

Next I asked Dr. Litchman if he could tell us about the two populations receiving the gene therapy currently and other patients who may benefit. Manny said that the therapy is currently being given to newborns with the disease. The incidence of the disease is very low with 1 in 225,000 live births globally. This results in about 20 patients in US. There is a larger population of about 400 who have received hematopoetic stem cell therapy and are eligible now or in the future for gene therapy because their condition has deteriorated due to infections. The National Institutes of Health (NIH) has a current trial running for these patients. Five patients have been treated and they are enrolling more patients and currently have a wait list.

I followed up by asking Manny what are the next steps for this therapy. He said that they will complete the technology transfer of the cell processing from St. Jude to Mustang’s cell processing facility in Massachusetts. It is a state of the art processing center run by Dr. Kniss. They will also continue the ongoing trial and will file for approval. At the same time, they will expand the number of sites sites for the NIH study to a multi center clinical trial.

I then switched gears by asking about their biomanufacturing approach. I began by asking Dr. Niss why they selected a lentiviral approach. Knut clarified that St. Jude selected lentivirus. He explained that it is well recognized that gamma retrovirus not the best approach for stem cell based therapies. He said we understand the integration of the lentivirus into the genome at a much higher level than the gamma retrovirus and gamma retrovirus integrates in a true random pattern where lentivirus has a defined integration pattern. For a therapy like this where you want durable outcomes, it is important to have stable integration into the genome so that the gene is expressed long term.

I then asked what were the biggest successes they found in manufacturing and what their plans are for scaling up in the future. Knut said that they don’t anticipate a problem because it is a fairly simple process. He explained that they isolate CD34 cells from the patient add the lentivirus and prepare the therapy for infusion. Capacity can be built up very quickly. He said that tehy are constantly scouting new technologies looking for the next technology breakthrough. In general, he said that cell and gene therapy products are now not the ultra rare niche technology. Technologies specifically developed for the needs of cell and gene therapy manufacturing have been developed and that has improved cell processing greatly

I asked what is next for Mustang. Manny said that they are looking for other in-licesning opportunities from the academic world of late pre-clinical or early clinical rare diseases that could take advantage of our cell processing facility. In the near term, they  have a rich portfolio in CAR T for oncology with 3 therapies for hematological malignancies and 3 for solid tumors. They will be  filing 2 INDs this year for two of those programs. One for AML and one for a multiple myeloma target.

I closed the interview by asking if there was anything else that they would like to add for our listeners. Manny said that they were very happy to have completed their capital raise and have at least two years of cash to execute on all their clinical programs. They are also grateful to their collaborators, St. Jude, City of Hope, Fred Hutchinson Cancer Center, and Nationwide Children’s Hospital.


Successful Methods for Perfusion Process Optimization



In this podcast, we interviewed Dr. Andreas Castan, Principal Scientist at GE Healthcare Life Sciences about the best methods for optimizing perfusion processes. This included a discussion of tools for media optimization and innovative cell separation techniques.

Show Notes

I began the podcast by asking Dr. Castan what makes perfusion a good manufacturing platform? He identified several reasons including short residence times in the bioreactor through adding fresh media and removal of spent media and as a result is perfect for unstable molecules. Perfusion permits processes to be run with high volumetric productivity by maintaining high cell densities for a long period. With perfusion there are many opportunities for process intensification and it is a good way to quickly get started with production, provided you have a good batch media that cells like.

Next I asked if there were any specific product types or situations that are an especially good fit for perfusion? Andreas described three primary areas – unstable products, integrated continuous manufacturing platforms and process intensifications for removing bottlenecks in manufacturing. Examples would be cell bank manufacturing to create high density cell banks, seed train to remove steps or produce high viable cell density inoculum or hybrid processes of both perfusion and fed batch technologies.

We then discussed the importance of optimization for perfusion processes and the various approaches for optimization. Andreas said it is important to optimize processes to gain the product quality and the process economy that you are aiming for. Cell culture media is the most important thing to optimize in perfusion culture. In order to achieve good throughput for your optimization it needs to be performed in a scale down model then results must be verified under bioreactor conditions.

Then I talked to Andreas about a recent poster he authored and presented at ESACT. The poster outlines development of perfusion specific media and designing medium that supports low cell-specific perfusion rates. I asked him to explain what cell-specific perfusion rate (cspr) is and why a low cell-specific perfusion rate is important? Andreas explained that cell-specific perfusion rate is the volume of media added per cell per day. If you assume your medium supports a cspr of 50 picoliter and you want to run the process at 30 million cells/mL your volumetric perfusion rate would be 1.5 bioreactor volumes per day. If you take the same medium with 100 million cells/mL you would need a volumetric perfusion rate of 5 bioreactor volumes per day, which is not feasible for production. A medium with a better depth of cspr of 10 picoliter at a cell density of 100 million cells/mL would have a volumetric perfusion rate of 1 bioreactor volume per day, which is feasible for production.

His team investigated screening methods to develop medium with a very low cspr. They started with basal media, then screened different feed solutions and Cell Boost feeds in batch mode. The Cell Boosts with a positive impact were taken to a DOE study in spin tubes that replicated perfusion conditions, then promising formulations were moved to perfusion processes in WAVE or the XDR bioreactors.

Poster – Perfusion media development for scalable processes

Next I asked him if you can turn a fed-batch medium into a perfusion medium? He said yes, transferring a fed batch media to a perfusion process is a simple process. He lays out the methodology in the previously mentioned poster and they have done this for two cell lines and three fed-batch media and feeds. The methodology is also described in the Biotechnology Process Journal Article, “Repurposing fed‐batch media and feeds for highly productive CHO perfusion processes.”

I followed up by asking how did the methodology work in the case study presented in the poster? Andreas said it was a very fast method with batch screening taking one week, the DOE study took two weeks and within one month they had the composition for a perfusion media that fit the desired clone. They tested two media developed using this process on their internal Herceptin producing cell line and for both media they reached cspr below 20 picoliter. They were able to reduce one media to only 7 picoliter. The cell specific productivity for this process was comparable to what they saw in fed-batch.

We then discussed another approach to optimizing perfusion processes, optimizing the equipment used in process. In another poster presented at ESACT, Andreas presented the use of hydrocyclones for cell separation. I asked him to describe how hydrocyclones work and why they make an attractive alternative to traditional cell separation devices? He said that hydrocyclone devices are comprised of cylindrical and conical parts that allow centrifugal separation provided by feed suspension introduced tangentially at high flow rates into the device. The absence of rotors or moving parts make it an interesting separation alternative to perfusion in long term operation. A simple device that is not prone to clogging as is usually seen with filtration devices

Poster – Hydrocyclone for mAb production in a perfusion single-use bioreactor

I then asked what his experiences were with the hydocyclone in the case study? Andreas explained that they investigated separation efficiencies at different flow rates and different flow concentrations and found that separation efficiencies of 70-80% could be achieved with the current hydrocyclone. This test was run using a perfusion process in an XDR 50 bioreactor at 50 million cells/mL for more than 2 weeks in very stable conditions. It was a very successful test of the device.

I summarized by asking if companies are limited in time and/or resources for optimizing their perfusion processes, what would he focus on first? He said media should be focused on first because as explained earlier a low cspr is key to reaching high volumetric productivity, low volumetric perfusion rates and high product concentrations, all of which result in good process economies. Next you could think about how you could use perfusion to reduce process scale.

Lastly, I asked if he had anything else that he would like to add for our listeners. He said that before moving into perfusion you should ask yourself what makes the most sense for your process. You need to think about your infrastructure, prerequisites, platform, and previous knowledge. Then you can decide where perfusion makes the most sense in your process. In cell bank manufacturing, seed train, or production bioreactor. Media is the most important factor to work with and it is not difficult to use existing media and cell boosts to develop perfusion media that fits your clone.