Cytiva - Exploring the potential of technology and data to advance gene therapy

EXPLORING THE POTENTIAL OF TECHNOLOGY AND DATA TO ADVANCE GENE THERAPY

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EXPLORING THE POTENTIAL OF TECHNOLOGY AND DATA TO ADVANCE GENE THERAPY

In the wake of a major integration, Emmanuelle Cameau, Strategic Technology Partnership Leader for Gene Therapy at Cytiva divulges how the company is transforming the gene therapy field.

ON MAY 1, 2023 PALL LIFE SCIENCES AND CYTIVA –BOTH PART

OF THE DANAHER CORPORATION – UNITED TO OPERATE UNDER THE CYTIVA NAME.

oining forces forged an even stronger global provider of services and technologies that focuses on advancing and accelerating the development, manufacture and delivery of therapeutics. With a wealth of technical expertise and talent, a broad and deep portfolio, and exceptional service, Cytiva helps researchers and biopharma advance therapeutics at every stage: from discovery to delivery. The company is now even better positioned to serve its customers as one

standalone operating company in the Danaher Corporation.

Here to tell us more about how Cytiva is wielding technology, analytics and strategic partnerships to serve patients and advance the gene therapy field is Emmanuelle Cameau, Strategic Technology Partnership Leader for Gene Therapy, part of the Genomic Medicine Chief Scientific Office team at Cytiva.

Emmanuelle’s role involves wearing many

hats. She is a thought leader in gene therapy and works closely on the content distributed by Cytiva related to the field. This extends to a commercial role, where Emmanuelle consults with customers about the various solutions and products offered by Cytiva. Lastly, Emmanuelle supports the marketing and R&D departments because she regularly engages with C-suite executives and researchers, so she understands their needs or challenges, which she then feeds

back into Cytiva’s development strategies so the company can serve current and prospective clients even better.

In addition, Emmanuelle embraces the opportunity to be involved in industry groups, such as the BioProcess Systems Alliance’s Cell and Gene Therapy committee, the International Society for Cell & Gene Therapy’s Process Development and Manufacturing ERM (Emergent Regenerative Medicine) committee, and the

Bespoke Gene Therapy Consortium, representing Danaher in the manufacturing sub-team.

How can the industry utilise technology to drive down the COGS to better serve the patients?

This is one of the emerging topics in the gene therapy field that features prominently in Emmanuelle's work.

“Lately, there have been some great approvals of gene therapy drugs,” Emmanuelle says. “For instance

the latest has been Hemgenix, coming in at a record cost of $3.5 million per dose.”

While this may seem expensive for a single-dose treatment, Emmanuelle explains that when you balance this against the total costs of treatment or courses of therapy this will replace – which can cost closer to $20 million – it is actually an affordable advancement.

But Emmanuelle identifies that the problem is even though these treatments might be cheaper in the long run, they are still too expensive for public providers or healthcare systems to absorb in practice if they were rolled out to all the people needing these solutions.

However, by focusing on the R&D, manufacturing and quality control processes, particularly by

“LATELY, THERE HAVE BEEN SOME GREAT APPROVALS OF GENE THERAPY DRUGS”

leveraging new technologies and refined operational procedures, Emmanuelle insists that solutions providers like Cytiva can help the industry drive down the total COGS.

“There was a recent webinar by Clare Blue at Biogen where she states that only 2 per cent of the products manufactured actually goes to a patient because the rest is used for quality control, stability studies, etc,” says Emmanuelle. “So there's clearly some improvement that needs to be made on the quality control assets and methodology to try to reduce the number of samples that are needed at each step so that we still keep the safety and the controls that are needed but with a lower quantity of samples.

“When I’ve talked to some key opinion leaders, I’ve asked how much we need to decrease the cost to get it more affordable and most people align on the fact that we need to be in a factor between 10 and 100 times cheaper.

“Within the manufacturing process, for example, if you look at the downstream for adeno-associated virus (AAV) vectors, the yield is usually around 25 per cent. This is not a great yield. Some customers have reached 40 per cent but if you take 25 per cent as a worst-case scenario, it becomes clear that a yield in the downstream of 100

“WE INTERACT WITH POLYPLUS IN COLLABORATION, FOR INSTANCE WHEN WE USE THEIR TRANSFECTION REAGENTS TO GENERATE MATERIAL OR DO EXPERIMENTS THAT HELP US TOWARDS OUR THOUGHT LEADERSHIP WORK”

per cent is not realistic. But say we strove towards 80-90 per cent. It means that from 25 per cent you are increasing the yield (and therefore reducing the costs) by about three times – so with all the improvements we cannot reach the 10-100 times improvement I mentioned earlier by key thinkers needed to make therapies affordable and realistic.

“So the answer is to look at the upstream side, that is where the big levers are. So these might include improvements around the transient or transfection process, it's around getting better transient transfection reagents, better cell lines that

are higher producers and better plasmids because we know plasmid quality also has an impact on the productivity and quality of results.

“Last year Cytiva acquired CEVEC, which is a company that has developed the only real stable cell line on the market. Stable cell lines will probably coexist with transient transfection because they make a lot of sense in the development of large indication therapies, high dose therapies, but not so much when it's just a very low dose, low indication say if we have to do a 50-litre batch every year.

“It's not only a question of higher productivity. So for viral vectors and specifically for AAV it’s about finding ways to increase the full/empty ratio. Right now we have processes that have 20 per cent full/empty ratios or more, and some processes have 2 per cent full. But if we were able to get the same productivity, so not even getting better, but get more full AAV particles, we would be making important progress.”

“Cells are living organisms so it’s very often a black box and we don't completely understand what happens in viral vector manufacturing. We need more consistency and a better

Guillaume Freund, Phd. Scientific Support Manager, Polyplus support@polyplus-transfection.com

Gene Therapy innovators must deal with constant pressure to get their product(s) to the clinic as fast as possible. In this aspect, the speed and flexibility of transient transfection of plasmid DNA to generate functional recombinant AdenoAssociated Virus (AAV) particles are cutting edge advantages over other techniques such as stable cell line generation. Despite being widely used, triple transfection has often been overlooked and labelled as “challenging”. While timing is key in an ultra-competitive market, a growing number of companies realise the importance of establishing robust and highly optimised manufacturing bioprocesses to produce enough potent viral particles. Approximatively 1:100,000 assembled AAV capsids will display the desired clinical output in humans (1) emphasising the fact that viral manufacturers must invest resources to build efficient AAV vector production platforms. And this goal can only be reached by having a robust and consistent plasmid DNA delivery method in place in the upstream process. Allocating proper resources to finetune triple transfection can have a huge positive outcome on the amount of functional viral particles produced.

The path to successfully reach clinical and commercial stages largely depends on the Process Development (PD) efforts deployed for AAV manufacturing, whether you outsource them to a Contract Development and Manufacturing Organisation (CDMO) or perform them in-house. The triple aim of such PD activities consists in:

Achieving highest viral titer

With highest viral particle functionality

Using an economically viable manufacturing process.

To reach this simply put yet ambitious goal, innovators need to develop a consistent and reproducible process. Plasmid DNA delivery is at the core of success since setting up a robust triple transfection step is often considered as the main challenge of upstream bioprocessing. Thus, it is critical to determine the best set of conditions for each specific AAV manufacturing workflow. There are many factors impacting the output of plasmid DNA delivery experiments to produce AAV vectors (Figure 1).

Figure 1: Non-exhaustive set of parameters to explore during optimisation of a triple transfection process for AAV manufacturing.

Hitchcock T, et al. Development Approaches to Adenoassociated Virus Production. BioProcess International (2017). Navigating the Complexities of AAV Scale-Up and Manufacturing. Genetic Engineering & Biotechnology News (2020). Sanderson TP, et al. Scalability comparison between 50 and 500 liter stirred tank bioreactor for production of rAAV viral vector. Cell & Gene Therapy Insights (2021). Hebben M & Nyamay’Antu A. Optimizing the AAV Transfection Process in Suspension Cells. BioProcess International (2021).

Each variable should be carefully studied during PD to identify the best performing transfection conditions and finetune the design space to operate in to target appropriate Critical Quality Attributes (CQA) of the process. Until now, most of the work done to improve rAAV production in the field often relied on optimising a single factor at a time. However, this approach is no longer favored because of (1) the multiplicity of factors contributing to rAAV generation, and (2) how these variables can interact. Design of Experiments (DOE) is now widely used to tackle this complexity by enabling to optimise each factor and ultimately determine a unique set of parameters for each specific viral vector manufacturing process. DOE approach allows to screen and optimise many factors, but you first need to identify and rank your drivers to draw clear experiments objectives as optimal transfection parameters cannot be determined in one single experiment.

Also, best performing transfection conditions for one serotype are not necessarily optimal for another – even when producing two different vectors using an identical tissue culture system. This really underlines that spending resources in early stage to tweak the workflow is key. Sometimes less is more. For example, when using the appropriate transfection reagent and conditions, reducing the total DNA amount can improve cell viability and increase overall rAAV particles quality for optimal potency in vivo without compromising on productivity.

Thereby focusing on VG titer alone might lead to back-andforth optimisation between USP and DSP because of poor quality viral particles. You cannot spread yourself too thin when you want to quickly get your product into the clinic. Each PD experiment matters, we will never say it enough: transfection conditions and cultivation parameters need to be optimised for each unique rAAV vector and cell culture setup. And Polyplus offers everything needed to do so.

Appropriate outputs are required to characterise any manufacturing process such as the physical titer expressed as Viral Genomes (VG) per mL of crude harvest. This readout is universally used for rAAV vector titration. VG copies are measured by quantitative PCR (qPCR) or droplet digital PCR (ddPCR), both techniques allowing to screen and select best performing experimental conditions in a sensible timeframe. While relatively quick and easy to determine, the physical titer is not always directly correlated with the final potency and quality of the recombinant vector produced. Thus, even if reaching the highest physical titer remains of primary importance when conducting upstream PD activities, the number of VG produced should not be used as sole criterion to cement best performing transfection conditions moving forward. Assessing functionality of produced viral particles is critical to align both UpStream Processes (USP) and DownStream Processes (DSP) objectives when developing a novel rAAV production platform. In this way, quantifying the proportion of full particles in the crude harvest while measuring their infectivity is required to confirm that the rAAV production method can consistently produce enough potent vector. Infectivity or functionality is usually determined with an in vitro assay using the closest cell type possible to the one targeted in the clinical application (2).

For many years now, PEIpro® is the leading DNA transfection reagent used in viral vector manufacturing processes for Gene & Cell Therapy products. PEIpro® has proven its efficiency, robustness and scalability in large-scale manufacturing of preclinical and clinical batches of rAAV. Abeona Therapeutics exemplified these characteristics by demonstrating the scalability of their transfection-based production process from 50 L to 500 L stirred tank bioreactor3. However, 500 L scale is just the beginning. To keep increasing productivity while easing the scale-up of bioprocesses to even larger bioreactors, Polyplus launched the new generation reagent FectoVIR®-AAV to revolutionise transfection-based rAAV manufacturing platforms: more titer, more flexibility, more scalability, more of everything can be expected when using FectoVIR®-AAV for your rAAV transfection process in suspension cells. Optimising transfection parameters with FectoVIR®-AAV based on a DOE approach can lead to unmatched yield improvements compared to standard triple transfection experiments using commercially available PEI. By combining LogicBio Therapeutics proprietary plasmid technology, Exothera's scale-up capabilities with our FectoVIR®-AAV reagent that ensures scalability of transient transfection, we recently demonstrated consistent high rAAV-8 titer yield, irrespective of scale from shake flask to 2000L scale bioreactor using Pall corporation Allegro STR system.

Polyplus’ unique transfection technologies supports continuous PD efforts deployed by viral vector innovators to make AAV manufacture affordable. Superior reagents such as FectoVIR®-AAV combined with DOE is most probably the best approach to optimise triple transfection experiments and in fine unlock the potential of each specific viral vector manufacturing process. All of this to ensure that each scientist successfully optimises their viral vector production process to ultimately bring Gene and Cell Therapies to market faster.

understanding of the cells and to find levers around higher productivity but also higher full/ empty ratios.”

How is the company deploying analytics technology development to assist viral vector manufacturing?

At Cytiva there are development and innovation projects underway to improve the use of analytics to bring solutions to viral vector manufacturers. But one of the perks of being part of such a large corporation as

Danaher is that Cytiva can benefit from the wealth of expertise and knowledge across its parent company’s entire portfolio.

For instance, there are companies like Beckman Coulter and Sciex that are dedicated, high-tech analytical providers.

“The big gap or question around this topic is that, currently, we don’t have many tools available to characterise and measure the ways we can improve viral vector manufacturing,” explains Emmanuelle. “For example, when

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we undertake what we call polishing steps or full/empty separation, we do not necessarily have the tools to differentiate one species from the other in a systematic way. And this is the next step for us as technology developers together with the drug developers – we are looking to build the tools to understand how our products help purify and separate these different species to go a bit deeper than just focusing on the full/ empty ratio alone.”

opportunity to forge strategic tech relationships with the goal of facilitating innovation and furthering the cell and gene therapy fields.

One of Emmanuelle’s roles at Cytiva is to set up collaborations to generate data together.

She highlights Cytiva’s ongoing collaboration with Virica Biotech.

How does Cytiva forge strategic tech relationships and facilitate innovation to further cell and gene therapy?

On the topic of building new tools, technologies and solutions, Emmanuelle highlights the importance of collaboration with innovative start-ups that are addressing some of the pressing challenges in the gene therapy sector.

But beyond collaboration with start-ups, Cytiva embraces the

“Virica produces viral sensitisers (VSEs) and they had some data on using VSEs to increase the productivity of viral vectors,” says Emmanuelle. “So we set up a collaboration with them to test their technology on human embryonic kidney cell line producing AAV 8. The goal is to see if VSEs drive productivity in a dynamic system not just flatware. We are also looking to see if they can improve full/empty as that’s the big lever but also see how they are cleared through the

“WE SET UP A COLLABORATION WITH VIRICA TO TEST THEIR TECHNOLOGY ON HUMAN EMBRYONIC KIDNEY CELL LINE PRODUCING AAV 8. THE GOAL IS TO SEE IF VSES DRIVE PRODUCTIVITY IN A DYNAMIC SYSTEM NOT JUST FLATWARE”

downstream as it’s a concern to potential customers.”

But Cytiva not only forges relationships to gather data, but to scale its operations as well.

Here Emmanuelle lauds Cytiva’s collaboration with Andelyn Biosciences, a contract development manufacturing organisation (CDMO).

“Andelyn is equipped with the relevant equipment from Cytiva’s, and we have a process development service – a fee-based offering from three labs in the UK, USA and Asia. We have worked with Andelyn on helping them scale up their upstream and downstream process, including process improvements that can make a difference at a large scale. It was a mutually beneficial collaboration

“WE HAVE WORKED WITH ANDELYN ON HELPING THEM SCALE UP THEIR UPSTREAM AND DOWNSTREAM PROCESS, INCLUDING PROCESS IMPROVEMENTS THAT CAN MAKE A DIFFERENCE AT A LARGE SCALE. IT WAS A MUTUALLY BENEFICIAL COLLABORATION”

Driving Client Value for Viral Vector Manufacturing Through a Platform Approach

Andelyn Biosciences is a Cell and Gene Therapy CDMO with 20+ years of experience manufacturing Advanced Therapies from its origins at Nationwide Children’s Hospital.

Driven by our mission of bringing more treatments to more patients, Andelyn offers a platform approach for suspension and adherent manufacturing that provides value based on three key cornerstones:

1. Cost Savings

• High yielding, scalable and high percentage of full capsids drives manufacturing costs down coupled with improved safety profile

• Assurance of program success through process experience and a track record of reproducibility and operational efficiencies

• Reduced upfront investment in Process Development Activities

• No Cell line licensing fees for manufacturing until BLA

• Continuous carrying of cells – no MCB/WCB manufacturing upfront costs

2. Expedited timelines

• Fully integrated CDMO from MCB/WCB generation to plasmid manufacturing, vector production and extensive in-house analytical capabilities

• Well-characterised platform reduces process development and analytical development activities scope and timelines

3. Reduced risk from IND to Commercial

• Ph1 to Commercial occurs in same site with no need for tech transfer

• Platform approach drives seamless transfer from Pre-Clinical to Commercial through process harmonization

• Nimble and scalable capacity to 2,000L

Scale

Maintaining Titer across multiple scales

because some of the work they did on the design of experiments and optimisation of their processes have transferred to us and we then scaled that up even further.”

As with any company, supplier relationships are key and here Emmanuelle discusses Cytiva’s close relationship with Polyplus – a leading upstream solutions provider that supplies Cytiva with transfection reagents and plasmids.

“We interact with Polyplus in collaboration, for instance when we use their transfection reagents to generate material or do experiments that help us towards our thought leadership work. We also engage with each other to support shared customers and to generate joint thought leadership content.”

What are some of the main opportunities or obstacles in the gene therapy industry and how is Cytiva leaning into them?

Emmanuelle describes the sheer volume of drugs that are in the clinical and late-stage clinical phase right now.

“We have shown we can make viral vectors and get them approved by the regulatory bodies and balance safety with efficacy,” explains Emmanuelle. “But they are still very expensive. So now we have to manufacture them with better, newer tools using analytics to make this process more efficient and better predict when we are developing a process to reduce the R&D costs that go into the therapy cost, so the therapies go into the clinic faster.

“There are some cases where therapies can be fast-tracked if the indications are incurable and depending on the country. But we need to be working together with the regulatory bodies so they can keep up with our new solutions. We now have all these different tools amongst mRNA, saRNA, viral vectors, cell therapy, gene-modified cell therapy, oligonucleotides etc, that form what we could call a scientific toolbox. But now scientists have to figure out what is the best tool for what problem, to cure specific diseases. This will be

a revolution because it will allow us to develop the best drugs or therapies for the specific diseases, optimally and effectively.”

What are the principal ambitions and growth plans at Cytiva in 2023 and beyond?

The ambition at Cytiva is to be the biggest supplier in the biotech industry, specifically within the genomic medicine industry.

With the combined Pall Life Sciences and Cytiva portfolio, Emmanuelle says, “We can really answer the customer needs from the early stage of the process to the final stage of the process for delivering therapeutics, regardless of scale, including process automation and/or single-use products.

“Our goal is to continue building on this prominent position while helping and advancing medicine – it’s our core driver to bring these lifesaving and life-improving therapies to people around the world.”

For more information about Cytiva, visit cytiva.com.

A NEW CHAPTER

Emmanuelle is thrilled about the recent integration of Pall Life Sciences and Cytiva.

“We’ve been working on this for years and it’s finally here and I can hardly believe the day has come to pass. But we believe it was the right thing to do and it’s pointing us in the right direction to help our customers and advance genomic medicines.”

EVENTS

Emmanuelle has her fingers crossed that she will be presenting Cytiva’s collaborative work with Virica towards the end of the year in Dublin at the Cell and Gene Therapy Manufacturing & Commercialization Europe conference.

She also invites readers to attend her Cytiva Live 30 webinar on June 13, 2023.

COLLABORATION FOR PROGRESS

Relishing the chance to advance medicine, Emmanuelle embraces opportunities for collaboration and continuing her work with industry thought leaders to give back to the world, an endeavour she finds incredibly motivating and inspirational.

Connect with Emmanuelle