Overcoming Bioprocessing Bottlenecks By Combining Adherent And Suspension Systems

Microcarriers, support matrices that enable the growth of adherent cells in suspension bioreactors, have the potential to simplify scale-up for challenging cell therapy applications. In a recent internal study, Waisman Biomanufacturing was able to demonstrate comparability for a new manufacturing platform combining bioreactors and microcarriers using mesenchymal stromal cells (MSCs). Using bone marrow MSCs as a model system, Waisman, utilizing bioreactors in combination with Corning Synthemax® II Microcarriers, was able to demonstrate comparable population doubling times (DT) when compared to typical adherent approaches (Figure 1) . Harvest densities and yield in 125 mL and 500 mL spinner flasks was also comparable 2D (data not shown). Results in pilot 3L bioreactors showed strong feasibility and cell viability (data not shown), but did show an increase in doubling time (DT), as shown below, suggesting more optimization at pilot bioreactor scale would be valuable prior to further scale-up.

Bone marrow MSCs are limited in their scalability due to their tendency toward cellular senescence when in vitro. Yet the depth of scientific understanding that exists for both the industry and within Waisman Biomanufacturing, made MSCs an ideal model system to demonstrate how microcarrier systems can complement traditional adherent approaches. This approach, blending adherent and suspension systems, has the potential to facilitate improved scale-up for a range of cell therapy modalities, including tumor-derived cell lines, pluripotent stem cells, and MSCs, including those derived from more novel sources, such as induced pluripotent stem cells (iPSCs).


Improving Scalability for Adherent Cells Through Microcarrier Technologies

Typical production for adherent cell applications begins with media screening followed by evaluation of a cell line’s growth properties inside small, single-layer T-flasks. This initial cell culturing can offer valuable insight into growth rates, seed densities, and optimal cell harvest approaches. This is often followed by scaling to a multilayer Corning CellSTACK® culture chamber or higher-capacity vessels such as the Corning HYPERStack® vessels, depending on an application’s needs for subsequent animal studies or clinical trials. For bone marrow MSCs, the process is straightforward and its ultimate scale dependent on the number of passages cells can undergo before they senesce. Although cells tend to grow fairly consistently within stacked vessels, this approach can be more time and labor intensive and has greater risk to maintaining sterility, particularly as an application scales. This is often due to the manual manipulations required for these systems, compounded across multiple vessels – 10 or 15 vessels, for example, to produce the same volumes as a single bioreactor. Yet the advantages of growing certain cells in a more natural adherent environment make retaining their advantages while improving their scalability imperative for many complex therapeutic assets.

To address these challenges, Waisman set out to establish a platform workflow that could enable cell expansion using Corning microcarriers. In partnership with one of Corning’s field application scientists, Tom Bongiorno, Waisman developed a workflow that includes initial screening in six-well plates to confirm cells’ propensity for binding to a particular microcarrier, as well as whether cells could be detached from the microcarriers. From there, Waisman worked to scale its process into Corning spinner flasks and finally, into a three-liter bioreactor. The goal was to demonstrate efficient expansion of MSCs on microcarriers, compared to traditional planar culture. Having achieved comparability for bone marrow MSCs in the microcarrier system, Waisman plans to offer expansion for various cell types well-suited to this approach, particularly stem cells. Its applications could also extend beyond cell therapies to derivative products, such as exosomes or secretome-derived products, which are seeing increased interest across the industry.

Improving Control and Reducing Manual Operations With Microcarriers

The potential benefits of combining adherent and suspension systems by way of microcarriers are manifold for anchorage-dependent cells – growing cells within a single bioreactor can afford operators improved control over media parameters, nutrients, and waste streams, enabling a more optimal, scalable process. This represents a game changer for adherent cells capable of expanding indefinitely, circumventing more labor-intensive approaches or solutions requiring extensive automation and robotics to make feasible. Using microcarriers to enable suspension for stem cell therapies also has potential for improving their scalability, enabling these cells to better maintain their potency and differentiation capacity.

For organizations looking to support a larger trial, move toward later development phases, or invest early in evaluating the scalability of an application, this new workflow may be pivotal in determining an asset’s eventual GMP production approach, according to Brian Dattilo, director of business development at Waisman. “I have confidence we can execute this workflow to evaluate if cells will adhere to a microcarrier that is suitable for GMP production, if we can get them off of the microcarriers, and do some economic analysis on cost of goods and economies of scale, to determine feasibility,” he said.

Importantly, Waisman was able to demonstrate comparable growth parameters and nutrient and waste analysis between a small-scale, 125-mililiter spinner flask and three-liter pilot bioreactor, despite differences in agitation for both systems that can affect gas transfer and fluid dynamics. “Despite the differences in some of the physics between the smaller scale and the three-liter, we saw very consistent growth parameters and other factors that make us confident in scaling beyond three liters,” Dattilo said. “There are always technical challenges that need to be overcome, but the differences scaling up withing bioreactor series aren’t as dramatic as transition from spinner flasks to small bioreactors.”

Leveraging Technology and Supplier Expertise for Better Production

The optimization needed to establish Waisman’s new platform required an intensive step-by-step process across each scale, from testing cell surface attachment in multi-well plates to analysis across key parameters, such as cell-to-microcarrier ratios, at both the spinner flask and bioreactor scales. Optimizing processes involving microcarriers can be a complex undertaking – microcarriers such as those offered by Corning can come with various surface coatings, for example; moreover, though Waisman elected to do the majority of its optimization using traditional polystyrene microcarriers, they are also looking to explore Corning dissolvable microcarriers, which may offer suitable applications a simpler, more efficient cell harvesting process. “For example, some customers may use microcarriers and suspension bioreactors as part of their seed train,” said Tom Bongiorno, field application scientist for Corning. “The dissolvable microcarriers are more costly than the polystyrene, so there’s a tradeoff there, but with that cost comes a reduction in labor – it takes a lot more labor to harvest the cells off of the polystyrene. It also does scale better to get a high cell recovery off of the Corning dissolvable microcarriers compared to the polystyrene.”

Ultimately, finding a CDMO willing to perform the process optimization necessary to accelerate complex biopharmaceutical development is crucial for companies looking to succeed in a highly competitive landscape. CDMOs that demonstrate a willingness to work with their suppliers to address bottlenecks are particularly valuable, as much of the consistency and scalability achievable for these processes hinges on the technologies and instruments designed to support them. Finding a manufacturing partner who has the expertise and the dedicated personnel, like Corning’s Field Application Scientist team, to be able to work iteratively toward achieving greater scale for these newer modalities can help companies realize more efficient, cost-effective production, paving the way for improved patient access across the advanced therapy space. Likewise, partnering with a CDMO like Waisman Biomanufacturing can afford companies deep expertise and flexibility in optimizing their development and scale up, enabling a more streamlined, accelerated path to commercialization.

About The Contributors

Brian Dattilo, Ph.D., is the Director of Business Development at Waisman Biomanufacturing. Dattilo earned his Ph.D. in biochemistry from Vanderbilt University, where he focused on recombinant protein production, purification, and analytical characterization. Prior to Waisman, Dattilo served as a program manager at the Biomedical Advanced Research and Development Authority (BARDA), where he was responsible for a multimillion-dollar development budget supporting novel platform technologies and their application to vaccine, biological therapeutic, and diagnostic development for pandemic influenza and biodefense applications. Since joining WB in 2012, he has led client interaction, technical product development plan development, and project budgeting and cost estimation, as well as built business cases for new platform investment.

Tom Bongiorno, PhD, is a field application scientist at Corning Life Sciences, covering the central United States. He has expertise in stem cell therapy, bioprocessing, media development, and cryopreservation. Bongiorno holds a PhD in bioengineering from the Georgia Institute of Technology and a BS in mechanical engineering from the University of Notre Dame.

About Corning Life Sciences

Corning Life Sciences is a global, leading manufacturer of products for growing cells, bioprocess manufacturing, liquid handling, benchtop equipment, and glass. Corning strives to improve efficiencies and develop solutions that enable researchers to harness the power of cells to create breakthrough innovations. Corning supports research for several application areas including core cell culture, bioprocess, cancer research, primary and stem cells, drug screening, cell and gene therapy, disease modeling, lab automation, and more. Learn more at www.corning.com/lifesciences.

About Waisman Biomanufacturing

Waisman’s team of cell culture specialists have a broad range of experience that enables us to optimize the culture parameters unique to each product cell line. Working in close contact with each client, we develop a cell culture process and cell banking program that meets cGMP/ICH requirements. Our facility houses five cell therapy suites that are specifically designed for the cultivation of mammalian cells for use in cell therapy applications including production of master and working cell banks for biologics production and cell and gene therapies. To learn more, visit us at www.gmpbio.org.