“This has been a 20-year effort among four labs and multiple highly talented researchers,” Andrew F. Stewart, MD, director of the Diabetes Obesity and Metabolism Institute at Mount Sinai told GEN.

The new study, titled, “Harmine and exendin-4 combination therapy safely expands human b cell mass in vivo in a mouse xenograft system,” was published in Science Translational Medicine.

Diabetes is a worldwide medical concern for more than 500 million people, nearly 10% of all adults. Current diabetes treatments address the symptoms of the disease, replacing insulin or increasing the body’s ability to utilize insulin. However, these treatments do not increase beta cell numbers, leaving a significant gap in reversing diabetes.

Stewart and his team in New York with California collaborators, including Adolfo Garcia-Ocaña, PhD, chair in gene regulation and drug discovery research at City of Hope, strategized that the best way to treat diabetes is to reverse the fundamental cause of the disease. “Beta cell deficiency underlies diabetes treatment strategies such as pancreas transplant, islet transplant, and new attempts at growing new beta cells from human stem cells,” remarked Stewart.

Together, they designed and implemented the first drug screens, followed by cell culture experiments, and as presented in the current study, animal transplant and drug treatment models.

The initial drug screening experiments identified a small molecule called harmine that can induce beta cell replication, which was published in 2015. This is a natural compound found in some plants that works by inhibiting the enzyme DYRK1A. Five years later, Stewart’s team showed that any of the GLP1 receptor agonists currently on the market synergize with any DYRK1A inhibitor to produce much higher rates of beta-cell replication.

The researchers’ earlier studies demonstrated that inhibiting DYRK1A in beta cells could induce short-term cell proliferation in vitro. They utilized an advanced laser microscopy technique called iDISCO⁺ to visualize and quantify beta-cell survival, function, and proliferation. iDISCO⁺ makes tissue transparent, allowing for 3D visualization of immunolabeled tissues.

Taken together, these data suggested that beta cells might grow in response to a combination treatment of harmine and GLP1. Further experimentation showed that the beta cells could survive in culture and the current study moved from tissue culture to studies in mice.

The researchers next took immunodeficient mice, typically used as models for diabetes research, with implanted human beta cells, and administered a combined treatment of harmine and GLP1. The results were profoundly positive, showing up to 700% increase in human beta cells over the three-month experimental duration. Stewart added, “In addition to beta cell mass expansion, diabetes reverses rapidly.”

“This is the first time that scientists have developed a drug treatment that is proven to increase adult human beta cell numbers in vivo,” said Garcia-Ocaña. “This research brings hope for the use of future regenerative therapies to potentially treat the hundreds of millions of people with diabetes.”

Stewart told GEN, “We had hoped that we would observe a small increase in human beta cell mass in vivo. But we never expected to see a 300–700% increase. This should be more than enough to ‘fill up the beta cell tank’ and reverse most types of human diabetes.”

Concurrent with this study, the Mount Sinai team has also completed a Phase I clinical trial of harmine to assess its safety and tolerability in healthy individuals. “We had hoped that we would observe no drug safety issues with drug treatment, and this is what we observed,” Stewart said of the trial.

Collaborators are also working on developing next-generation DYRK1A inhibitors as part of their continued research with the aim toward imminent clinical trials.

“Our studies pave the way for moving [other] DYRK1A inhibitors into human clinical trials and it’s very exciting to be close to seeing this novel treatment used in patients,” Garcia-Ocaña said. “There is nothing like this available to patients right now.”

Stewart concluded, “The steady progression from the most basic human beta cell biology, through robotic drug screening and now moving to human studies, illustrates the essential role for physician-scientists in academia and pharma.”

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The work, which is published online today in Nature in the paper “In situ targeted base editing of bacteria in the mouse gut,” was a team effort by the scientists at Eligo, led jointly by Jesus Fernandez-Rodriguez, PhD, (Eligo’s VP of Technology), David Bikard, PhD, (Eligo co-founder and head of the Synthetic Biology Group at Institute Pasteur), and Xavier Duportet, PhD, (Eligo’s CEO and chairman).

Before this report, notes Bikard, “it was still an outstanding question whether it would even be possible to genetically modify a whole target bacterial population in an animal. There could have been fundamental barriers that would’ve made this impossible. But here we show that we can do it!”

Eligo’s advance, which combined research in both vector engineering and payload modification, is exciting on two fronts: it could open the door to new microbiome genome-editing therapeutic modalities. And it launches a microbiome-editing toolbox that has been previously unavailable.

This work is “ushering in a new era of microbiome engineering,” notes Rodolphe Barrangou, PhD, professor at North Carolina State University and editor-in-chief of The CRISPR Journal. “This proof-of-concept study is just not for E. coli or the mouse gut microbiome; it can be used much more widely, for all kinds of things, and can be deployed at scale.”

Brady Cress, PhD, principal investigator of microbiome editing technologies at the Innovative Genomics Institute at the University of California, Berkeley, agrees. Cress told GEN that this is “a massive step forward that opens the door to rewriting our microbiomes for optimal health.”

Tweaking, not altering

Duportet co-founded Eligo with Bikard a decade ago; the two friends were still in training when they had the idea for the company—Duportet was a graduate student at MIT, Bikard a postdoctoral fellow in Luciano Marraffini PhD’s lab at the Rockefeller University. (Marraffini and Tim Lu, MD, PhD, are Eligo’s other scientific co-founders.) Today, Duportet and Bikard are a dynamic duo—with Duportet at the helm of the company and Bikard a scientific advisor, who remain close friends while collaborating scientifically.

Current microbiome approaches are typically based on altering the compositions of the bacteria. The idea is to introduce bacterial species to change the balance (like probiotics) or to remove others. Eligo’s focus here was different. The idea is not to kill the bacteria but rather, as Duportet explains, to “inactivate its pathogenic potential and leave the bacteria in place.”

“If you are trying to target bacteria that has a niche,” notes Bikard, “completely removing it from the niche might be very challenging. Unless there is something else there to take its place, it will just grow back. So, it is a better strategy to disarm it, rather than kill it.”

Two-fold

Eligo’s new data are not the first to demonstrate editing of the microbiome in vivo, however. In May 2023, research from the Danish company SNIPR Biome was published in Nature Biotechnology in a paper entitled, “Engineered phage with antibacterial CRISPR–Cas selectively reduce E. coli burden in mice.” In it, the researchers identified eight phages (after screening a library of 162 phages) that delivered a CRISPR-based gene-editing payload that resulted in a reduction of E. coli in the mouse gut. In the SNIPR Biome study, the E. coli were killed by CRISPR.

It has been known for a decade—since Bikard and Marrafini’s Nature Biotechnology paper in 2014—that cutting the bacterial chromosome with CRISPR-Cas kills bacteria efficiently. But the first generation of gene-editing tools were not very efficient tools, notes Bikard. They could be used in a lab setting to make modifications but most of the bacteria would be killed in the process. Therefore, if the goal is to make modifications in vivo, and maintain the bacterial population, the first generation of CRISPR tools for bacteria “would not cut it.”

The game changed when base editing—a more precise form of genome editing developed in the Broad Institute lab of David Liu, PhD—entered the toolbox. Eligo worked to bring together their knowledge in genome editing and delivery to allow for efficient editing of bacteria without changing the composition of the microbial population.

When targeting E. coli strains colonizing the mouse gut, Eligo’s technology modified the target gene in more than 90% of the bacteria, reaching up to 99.7% in some cases. These modifications remained stable for at least 42 days.

Barrangou notes that the penetrance of the edits showed remarkable efficiency. “They are setting the bar,” he told GEN. “Being able to do it is one thing. But being able to do it with that kind of longevity and efficiency is practically important and sets the stage for new opportunities in the field.”

For Bikard, working out the gene editing was not as big a hurdle as the delivery. Eligo modified the site fibers on a phage chassis (from phage lambda) to target specific bacterial strains. The phage vector can be modified to target different bacterial strains or species with additional engineering.

Cress thinks of it as “a reprogrammable platform” for targeting different bacteria. That said, while this research provides an impressive blueprint using the most well-studied phage-bacteria pair, Cress notes that expanding it to other microbes will necessitate developing efficient genetic tools for non-model bacteria and a deeper understanding of the genetics and biology of less well-studied phages.

Another advance, Duportet notes, is that his team was able to demonstrate the same efficiency of editing using a non-replicative plasmid in the target bacteria. This is an additional benefit because they don’t maintain a transgene in the microbiome of the animals.

Long road ahead

The long-term goal at Eligo is to develop therapeutics—not necessarily for infectious diseases. The interest extends to those that would change the genetic content of the microbiome that alter a factor of host–focused diseases.

One example where this could be applicable is the delivery of a base editor to commensal intestinal E. coli that express the toxin colibactin, to inactive its mutagenic potential, therefore preventing the progression of human colorectal tumors.

But there is a long road ahead and challenges remain. One, notes Cress, is that this approach uses short-lived delivery of gene editing machinery to make gene disruptions, but other types of edits like gene insertions take longer to write (e.g. CRISPR-associated transposases) and thus will likely require different delivery approaches. Another point Cress considers is that “the type of edits made in this study could potentially revert through natural mutation, making gene removal a more durable solution than gene disruption.”

This study also raises new questions. Teasing apart the genetic network of the microbiome is in its infancy. Do researchers have enough knowledge to use this new tool?

“It’s important to have the genome editing tools,” notes Barrangou. But in the end, “what really matters is knowing what to target. Knowing what to target and what edit you want is part of the secret sauce.”

But Bikard reckons that this work will help answer some of those questions. This will be an extraordinary tool for researchers, he says, because it offers the possibility to probe gene function directly in the animal. He is excited to use it in his academic lab on the other side of Paris from Eligo’s base.

Duportet hopes that scientists will use the method and is happy to issue “a call for collaboration.” “We cannot work on everything, and we cannot find all the targets to edit,” he notes. “But we have the knowledge to design the vectors and the payloads to make it happen.”

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“With biosimilars, it’s cost that drives the market. Process optimization gives the best possible yield while having a minimal effect on potency and safety,” says Hausfeld who will be discussing how to fund biosimilar development at the Bioprocessing Summit in Boston next month.

As part of his talk, he will detail the optimization techniques that BioFactura has adopted to cut costs as, for example, intensified perfusion which, according to Hausfeld, uses more expensive culture media, but also produces higher yields.

The company has also adopted CO₂ stripping to maintain the pH of the bioreactor without adding bio-carbonates that can affect the health of the cells. “From what we can tell, the CO₂ doesn’t have any negative effects on the critical quality attributes of our product,” he says.

Role for metabolomics

Finally, they’ve analyzed cell lines with metabolomics to see how their productivity varies over time and, we’ve identified three supplements to improve productivity,” he says, adding that process optimization is best done after Phase I.

“Until Phase I, you tend to have a locked process,” Hausfeld explains. “Afterwards, you might realize how you can tweak without a major change that would raise the eyebrows of a regulator.”

Regulators look to make sure that a biosimilar product resembles the original drug. Too many variations, and they will expect costly clinical trials. Hausfeld also recommends that biosimilar manufacturers carefully record the steps they take to optimize processes during development. This, he says, can help companies more easily transfer their technology to a contract manufacturer for commercial-scale production.

“The complexity of tech transfer can be [a] major issue that people don’t always think about when developing drugs,” he says.

“There are nuances that occur during every bioreactor run, and the tech transfer process can take a long time and go astray if you don’t document what you do in a stringent manner,” he continues while also recommending that instructions sent to contract manufacturers be readable, understandable, and actionable, keeping in mind that there are lots of opportunities to ask questions and exchange information before beginning a manufacturing process.

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As a tool developer, “our mission is to make analytical tools easily accessible so the bioprocess scientists developing processes can perform key analysis themselves,” rather than having to hand off analyses to other labs, Graziella Piras, a senior director at 908 Devices, tells GEN.

The benefits for biomanufacturers accrue in the form of accelerated processes, lower analytical costs, and process intensification, culminating in reduced time to market.

For example, 908 Devices’ Rebel at-line cell culture media analysis device claims to reduce analytical process time from the conventional three to six weeks to 12 minutes. In the biopharma industry, such time savings add up to major money savings.

“One month of process development costs approximately $1 million,” Piras said, citing a 2020 study by University College London and AstraZeneca.

That study showed that to achieve an overall 12% approval rate for Phase I drugs, for material preparation alone, a biopharma company should expect to spend about $60 million from preclinical to Phase II and about $70 million for Phase III to regulatory review. For a 4 percent success rate—like that of drugs targeting Alzheimer’s disease—the costs increase 2.5-fold.

Therefore, she said, “If you can save a couple of weeks’ time, it matters.”

Real-time analysis matters

On the biomanufacturing side, the focus is on maintaining drug quality and efficacy. “We’re talking about living cells,” she stresses. Anything less than optimal conditions can reduce the quantity of drug produced. Even a small reduction can have “a huge effect on the cost of goods.”

To manage that, Piras advises real-time monitoring and analysis to pinpoint where and when optimal parameters vary, and to enable near-real-time responses. When Terumo Blood and Cell Technologies (a client of 908 Devices) implemented real-time monitoring to control glucose and lactate concentrations in cell cultures, sampling data points increased from one or two per day to as many as 720 per day, thereby providing deeper insights into the process. The risk of contamination also was reduced by eliminating the need for manual sampling.

It’s important for process specialists to not only have the data in real-time, but also to have the interpretation of that data immediately, she points out.

The challenge, Piras says, is that, “the industry is slow to adopt innovative solutions. In that regard, biopharma companies have a fear of being first. They are risk averse.”

That’s natural in highly regulated industries, but regulators, she points out—including the FDA—are eager to work with companies. The FDA wants to help developers adopt platforms that enhance development and manufacturing efficiency and improve drug safety and efficacy.

To reduce the risks, Piras advocates becoming involved in consortia, like NIIMBL, that bring together industry, academia, and innovative tool developers. By collaborating early on, they can advance biomanufacturing innovation.

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Today most purification methods are based on separating biopharmaceuticals from cellular debris, reagents, and nutrients present in the process stream. The aim is to pass large volumes of liquids through filtration systems as efficiently as possible.

However, for personalized medicines produced in smaller quantities the challenges are different, according to Dong-Pyo Kim, PhD, director of the Center for Intelligent Microprocess of Pharmaceutical Synthesis at Pohang University in South Korea.

“The personalized medicines industry needs new, super-efficient purification technologies because it handles complex biological molecules like nucleic acids, proteins, and cells that must be extremely pure for safety and effectiveness. A small impurity can result in significant immunological side effects.”

And there are other motivations for the development of more efficient downstream technologies, with cost reduction being the obvious example.

Kim tells GEN: “The purification process represents approximately 60–90% of the total cost involved in producing biotherapeutics. New approaches must be focused on boosting purity, cost reduction, flexibility in production scale, and fit smoothly with other manufacturing steps.

“Current technologies just aren’t flexible or precise enough for small, individualized batches, making it tough to keep consistent quality and meet regulations, which also pushes up production costs,” he says.

Microfluidics

Instead of using current tech—Kim and colleagues argue in a new study—personalized medicine developers should use systems designed to process smaller volumes—so called “microfluidics”—for downstream processing.

“Microfluidic technologies are a game-changer for purifying personalized medicines, specifically for those small population patients suffering from genetic and rare disorders because they offer high precision and scalability. They can handle small, customized batches efficiently, which is perfect for personalized treatments.

“These systems cut costs by using smaller quantities of reagents and minimizing waste. Plus, they provide high-resolution separation to distinguish closely related biomolecules and achieve great recovery rates, making the most of valuable therapeutic agents,” Kim says.

He also advocates combining microfluidics with automated artificial intelligence (AI) based monitoring, analytics, and modeling technologies.

“Combining microfluidic technologies with AI and automation systems brings a lot to the table, specifically for producing biotherapeutics and personalized medicines. AI can analyze real-time data from microfluidic processes to fine-tune things like flow rates and reaction conditions, making everything run smoother and more efficiently. It also uses past data to predict outcomes, helping us make better decisions, and keeping experiments on track.

“With automation in the mix, tasks like preparing samples and purifying substances become super precise and repeatable. This setup speeds up the whole development process by cutting down on manual work and analyzing data faster. It also keeps a close eye on quality, ensuring each batch meets high standards while managing costs better by optimizing how we use resources.”

Kim adds, “Overall, it’s a powerhouse combo that’s pushing the boundaries of biopharmaceutical processing.”

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In the introduction to Recent Advances in Bioprocess Engineering and Bioreactor Design, published earlier this year, Archana Vimal, PhD, assistant professor of bioengineering at Integral University in Lucknow, India, and her colleagues wrote: “Besides umpteen useful traits, bioprocess technology still needs to overcome a large number of hurdles and possess an advantage over other competing methods such as chemical engineering to be viable in any specific industrial context.”

To Vimal and her colleagues, a key bioprocessing hurdle is “developing the enabling technologies for industry to fully utilize the potential of contemporary biology and chemistry through synthesis and innovation.” As if that’s not enough, these authors want tomorrow’s solutions to also be sustainable and more.

As they noted, the bioprocessing industry should incorporate “manufacturing processes into environmentally acceptable and financially viable process concepts, rapid purification and monitoring of purification processes to produce products of high-quality, high-purity, and consistent output are some of the challenges that bioprocess technology must overcome.”

Techno-economic analysis

For one aspect of economic sustainability in bioprocessing, Satya Eswari Jujjavarapu, PhD, an assistant professor in biotechnology at the National Institute of Technology (NIT) Raipur in India, and Swasti Dhagat, PhD, a research scientist at SRISTI (Society for Research and Initiatives for Sustainable Technologies and Institutions), explored the economics of fermentation, including techno-economic analysis (TEA). As they pointed out, “many online tools are available that help in evaluating the feasibility of a production process.”

To provide an example, these authors noted that Michael Lynch, MD, PhD, the W. H. Gardner, Jr. Associate Professor of Biomedical Engineering at Duke University, developed a bioprocess TEA calculator.

“Techno-economic analysis connects R&D, engineering, and business,” Lynch explained in an article about his bioprocess TEA calculator. “By linking process parameters to financial metrics, it allows researchers to understand the factors controlling the potential success of their technologies.”

Perhaps the biggest challenge in bioprocessing is inherent in the name. It is a process. As shown here, many steps in bioprocessing need improvements, and tools exist to take on some of those challenges. Really, though, as bioprocessing jumps one hurdle, more will appear, often because of improvements in technology or evolving societal needs. It’s just the nature of the industry.

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The research, published today in Science in a report entitled “A molecular glue degrader of the WIZ transcription factor for fetal hemoglobin induction,” was led by Pamela Ting, PhD, associate director of hematology at Novartis Biomedical Research. Ting leads a team of 40 co-authors, including Jay Bradner, MD, PhD, the former president of Novartis Institutes for BioMedical Research, who left the company in 2022 and joined Amgen late last year. Developing small-molecule drugs for SCD has been a long-standing interest of Bradner’s, dating back almost 15 years to research he performed on histone deacetylase inhibitors in collaboration with Stuart Schreiber, PhD, and colleagues at the Broad Institute.  

Over the past decade or more, predating her own arrival at the company in 2015, Ting estimates that Novartis has screened some two million compounds in the search for a promising compound to take to the clinic. Along the way, the company briefly pursued its own CRISPR gene editing strategy, following a similar path to the approach that Vertex Pharmaceuticals took in developing Casgevy by boosting HbF production. (Although Novartis and collaborators published promising initial clinical data last year, the company decided to shelve the gene editing program.) 

Despite the remarkable clinical success of the CRISPR ex vivo approach—the first American patient in the Vertex trial, Victoria Gray, celebrated the fifth anniversary of her stem cell transfusion earlier this week—it is clear that this demanding (and expensive) ex vivo strategy will not help the millions of patients in Africa and beyond—a point that Ting and colleagues hit home in their report in Science.  

“Regrettably,” Ting and her co-authors write, “substantial challenges exist for [hematopoietic stem and progenitor cell] therapies to reach most SCD patients, who live in medically underserved communities and low and middle-income countries. Safe, efficacious, and globally accessible HbF-inducing medicines therefore remain an important unmet need.” 

Fyodor Urnov, PhD, director of technology and translation at the Innovative Genomics Institute, told GEN that the 2023 approval of Casgevy provided definitive clinical evidence that the strategy of upregulating HbF is “a safe and effective approach to resolve major symptoms of sickle cell disease.” The new work builds on the theme of modulating a transcription factor to upregulate HbF, but this time using a small molecule. “Even people who live and breathe CRISPR will agree that, on balance, such a small molecule could have a wider global reach than gene editing,” Urnov said. 

“There is a pressing need for oral agents that can more effectively induce fetal hemoglobin,” agreed hematologist Vijay Sankaran, MD, PhD, professor of pediatrics at Harvard Medical School. (Sankaran played a key role in cementing the HbF upregulation strategy to treat SCD 15 years ago.) He called the new Novartis report impressive and “exciting work and identifies a promising new target for fetal hemoglobin induction.” 

Screen time 

The Novartis study falls into a category known as “molecular glue pharmacology.” The researchers recognized that very small modifications to the chemical structure of a class of drug compounds that mediate targeted protein degradation (TPD) could profoundly affect the choice of protein for degradation. (A good example was work published by Novartis colleagues last year on the discovery of selective glue degraders for cancer immunotherapy).

“This was a big conceptual leap,” Ting explained. “You could do just a little bit of work around a fundamental structure and then broadly look for changes to the substrate selectivity.”

She added: “We decided, if this [chemical] library is capable of drugging a new class of transcription factors, then we should think about how we can apply it to an age-old problem that, at the root, is really a question of how do we regulate genes? And can we target transcription factors to regulate gene expression?” 

Ting’s team embarked on a screen of a library of almost 3,000 compounds that target cereblon, a component of the E3 ubiquitin ligase complex that is involved in targeted protein degradation. Using a cellular assay that detected the upregulation of HbF) in erythroblasts, Novartis researchers identified scores of candidate molecules before whittling down the list and focusing on “compound C” for further study.   

After mass spectrometry experiments showed that this chemical targeted the WIZ transcription factor, Ting’s group renamed the drug candidate dWIZ-1. The WIZ target was emphatically confirmed by CRISPR gene knock-out experiments. All told, these results suggested that WIZ was a previously unrecognized repressor of HbF expression. dWIZ-1 could recruit WIZ to the cereblon-ubiquitin ligase complex to trigger targeted protein degradation. The Novartis researchers continued experiments with a modified molecule termed dWIZ-2. 

The Science report is packed with multiple types of experiments—molecular assays, mouse and non-human primate models, protein modeling using AlphaFold, and X-ray crystallography. Part of the rationale, Ting says, “was to convince ourselves that by every measurement that we could think of, we could see a reproducible effect.”  

Some of the most important data in the report are results administering dWIZ-2 in a trio of cynomolgus monkeys. This in vivo study monitored the animals for 28 days and showed robust up-regulation of the γ-globin gene and HbF production in virtually all peripheral reticulocytes. “We saw almost pan-cellular HbF expression, which is very promising,” Ting said, and no signs of cytotoxicity. Curiously, one of the three animals was termed a non-responder. Ting calls it “the coolest monkey, because I feel like it must be telling us something and I don’t know what it is!” 

The Novartis team found evidence for WIZ binding directly at the β-globin locus, which was unexpected. “Seeing WIZ for the first time is quite surprising for such a deeply studied locus,” Ting said. “For whatever reason, the globin locus seems to be highly sensitive to the loss of WIZ.” What is striking, she adds, is that “there is relatively strong overlap with binding sites for BCL11A,” the transcription factor that Vertex successfully targets in its Casgevy cell therapy.

“That’s where we leave it in the paper, as a bit of a teaser, something that we hope that we can further study in the future and that others will be inspired to study.”  

Dealbreaker 

As Ting readily acknowledges, there are still many unanswered questions, including the specificity of the inhibition. WIZ naturally binds at many spots in the genome, including a dozen or more in the β-globin gene cluster. “For whatever reason, when we modulate WIZ, the effects are fairly moderate and fairly mild,” Ting said. And the results in the non-human primates bode well. “We hope with these new chemical probes that this is something that we can continue to study and understand in the long run,” Ting says.  

“I think the [Novartis] approach is promising, but with the current data it’s difficult to know what degree of HbF induction a human would experience,” said Vivien Sheehan, MD, PhD, associate professor and director of Sickle Cell Translational Research at Emory University School of Medicine in Atlanta. Sheehan, who first heard these results presented at the American Society of Hematology conference late last year, would like to see additional quantification data including HbF measurements using high-performance liquid chromatography. She also observes that “the lack of myelosuppression is promising for a potential combined therapy with hydroxyurea.”  

Sankaran cautions that the non-specific effects of targeting WIZ lead to expression changes of hundreds of genes in erythroblasts. “More studies are needed before and during human clinical studies,” he said. A rigorous assessment of the safety of systemic WIZ degradation will be needed. “That could be a dealbreaker,” Sheehan says. 

Understandably, Ting would not be drawn on a timeline for progress into the clinic, but her closing comments underlined her team’s belief in the program. “What we’re really focused on now is making sure that we’ve found a molecule that can really go all the way to patients,” she said. “We are highly committed to sickle cell patients around the world. We see the unmet need,” she said. “We’re progressing as quickly as possible, and we really have our eye on a medicine that will reach patients where they are.” 

Nevertheless, while a small-molecule drug should prove both more affordable and accessible in the countries where most SCD live, IGI’s Urnov cautions that access to drug therapies outside the U.S. is by no means guaranteed. For example, a decade ago, Gilead slashed the price of its drug Solvadi by almost 99 percent in order to provide meaningful access to hepatitis patients in Egypt. The Novartis study, Urnov says, offers “an important opportunity to think ahead to how to solve this challenge when and if a medicine based on this work is approved in the U.S.” 

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Vogel, who is speaking about sustainability at BioProcess International in September, says whether sustainability is about carbon footprint, water use, or other metrics varies by organization and roles within companies. “Sustainability currently means different things to different people—akin to the story of seven blind people touching different parts of an elephant.”

For some companies, sustainability can simply mean reducing carbon footprint. Here, he says encouraging employees to carpool can make more impact than altering manufacturing processes. Alternatively, companies might focus on water use as a measure of sustainability. “And [the biggest] issue is adopting single-use [bioreactors], versus multiuse,” he explains.

According to Vogel, depending on metrics and how organizations account for sustainability, it may not be clear-cut whether stainless steel equipment uses more energy and water than single-use plastics. “There are the optics of waste that requires landfill space and incineration, but multi-use equipment needs lots of water and energy [to clean],” he says.

No clear answer

Many studies say one is better than the other, he says. “I don’t have any [clear] answers, but single-use offers many advantages so, if you’re early in the process, [there’s no reason] not to do single use,” he continues. “Once you get to larger scales, you need to do a hard analysis with profitability and costs paramount, and sustainability added into that.”

Many companies consider sustainability less during lab-scale and Phase I manufacturing, which makes sense, he explains.

“It’s happening more in mature organizations,” states Vogel. “And companies going into Phase III with a CDMOs are asking about sustainability practices for their platforms.”

He recommends all companies no matter what stage of development or manufacturing consider sustainability seriously!  It will pay off for their bottom line and the planet!

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“The sterilization challenge for bioprocessors primarily revolves around ensuring that all bioprocessing equipment and products are free from any microbial contamination and endotoxins,” says Redmond. “This is crucial because even a small level of contamination can compromise the safety and efficacy of the bioproducts, such as pharmaceuticals, and can lead to serious health risks for patients.”

According to Redmond, bioprocessors face three key challenges in sterilization. First, she says, “Manufacturers need to consider sterilization during the design process of bioprocessing equipment to facilitate easier and more effective cleaning and sterilization.”

Risk assessment

Next, risk assessment must be considered. Here, she notes that revisions to the EU’s Annex 1 and ICHQ9 R1 “highlight the issues that pharma needs to address.” In addition, human error must be avoided. “The manufacture of aseptic products requires human intervention and good aseptic practices,” Redmond points out. “Many of the warning letters issued by the FDA site poor aseptic practices and understanding as the root cause of sterilization issues.”

Although Redmond states that “achieving complete sterilization of complex equipment and operational procedures is difficult,” various methods can reduce this challenge. For one thing, some advanced materials are microbe-resistant. Using these materials in bioprocessing equipment reduces the risk of contamination.

Where possible, equipment can be designed to include fewer hard-to-clean areas. Human error can be reduced with automated systems for cleaning and sterilization. Those processes should also be monitored continuously. “Using sensors and IoT technology to monitor the sterilization process in real-time can ensure that the required conditions are met throughout the process,” Redmond says.

Like the rest of bioprocessing, sterilization procedures keep improving. As Redmond notes, “sterilization methods are continually being refined to enhance their effectiveness, reduce processing time, and ensure that they are safe for both the products and the environment.”

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In a recent paper, UCD PhD student Laura Murphy, postdoctoral researcher Ciara Lynch, PhD, and associate professor David O’Connell, PhD, compared spent yeast culture media to the original rich biological media used to produce recombinant proteins in E. coli fermentation.

When growing E. coli in either medium, the team identified substantial depletions in levels of six elements and 18 of 20 amino acids. They noted that 98% of the starting levels of magnesium were consumed and found that two to five mM of magnesium were considered ideal for E. coli growth.

Magnesium ups growth and titer

Concentrations of sodium and magnesium were lower in the spent media, while levels of phosphorus, sulfur, potassium, and calcium were comparable to those of rich biological media, the scientists report.

Therefore, they added magnesium and sodium to replenish those levels. In that solution, doubling time for E. coli was 1.23 hours, compared to 2 hours for the depleted medium alone. Simply adding magnesium to the spent solution resulted in a 1.15 hour doubling time, while only adding sodium resulted in a 2.15 hour doubling time.

These results suggest “that magnesium supplementation is the most important elemental addition for successful E. coli growth in spent yeast media,” O’Connell’s team noted.

Additionally, the work showed that all amino acid levels were “significantly depleted,” save that of cysteine, which increased, and isoleucine, which declined only slightly. Supplementing amino acids, therefore, helps support the culture’s growth rate and productivity but has little impact alone.

The highest recombinant protein titer occurred when 2 mM of magnesium and 1% tryptone solution (to restore amino acid levels) were added to the depleted culture. The resultant cell dry weight was 2.7-fold higher than adding magnesium alone, and 1.6-fold greater than cells grown in in the original rich biological media. This, they reported, was “the most striking observation.”

This approach not only increased protein titer, but also decreased energy use.

Other researchers determined that the water-related impact of energy, for example, ranged between 18 and 80 kg per kilogram of antibody produced. Approximately 95% of the water’s process mass intensity is related to media. “Calculations indicate that this media recycling could save upwards of 800 kg of waste per kg of product,” O’Connell told GEN.

The team concluded, “Yeast spent media are viable media for reuse in feeding secondary E. coli cultures with minimal supplementation steps. These cultures can be highly productive.”

“The next steps,” O’Connell said, “will be for collaboration with industrial teams to assess this approach to valorizing yeast spent media post-clarification, and retrieval of recombinant biopharmaceuticals.”

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