Opportunities for BioManufacturing Sciences to Accelerate Upscaled Mesenchymal Stem Cell Manufacturing Technologies

Contributed by: Timothy R. Olsen, PhD - Sr. Scientist, Process and Product Development at RoosterBio Inc.

After the National Institute for Standards and Technology (NIST) identified upcoming challenges in the U.S. biopharmaceutical manufacturing landscape (see document here), the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) was formed to help solve these challenges through the formation of a public-private partnership between industry, government, academia, and non-profits. The goal of NIIMBL is to accelerate Biopharmaceutical manufacturing innovation, support the development of standards that enable more efficient and rapid manufacturing capabilities, and educate and train a world-leading Biopharmaceutical manufacturing workforce to maintain the United States’ global lead and competitiveness in this industry. NIIMBL will be leveraging a $70 million investment from NIST, with at least $129 million more in funds from private partners. One of the great aspects of this institute is that they are encouraging partnership between large companies and smaller or medium size companies (called Small to Medium Enterprises “SME”), which will be sure to bolster innovation and streamline commercialization and implementation of new technologies. Kelvin Lee, the NIIMBL Institute Director, did a fabulous job taking the lead on organizing the Consortium's first National Meeting, where members from the United States Congress, Directors from the Food and Drug Administration, and many executive level industry representatives were invited to speak about the importance of manufacturing sciences and the current challenges we are facing as an industry. I had the opportunity to represent RoosterBio as an SME at this inaugural NIIMBL National Meeting, and I gave a talk in the “Rapid Fire” SME Innovation Showcase, as well as presented some of our work on how we are radically shortening the development timelines of Biopharmaceuticals that include a stem cell-derived component.

Confluent hMSCs on Solohill microcarriers.
Image from RoosterBio Inc

Among the many relevant talks given throughout the day, one specifically caught my attention. In his talk titled “Key Process & Assay Challenges in Cell Therapy Development,” Greg Russotti (Vice President, Technical Operations at Celgene Cellular Therapeutics) laid out challenges in the upscaled manufacturing of human mesenchymal stem cells (hMSCs). To meet the pressing need for economical manufacturing of hMSCs at clinically- and commercially- relevant scales, researchers have turned to single-use bioreactor systems that have successfully been used to manufacture other biomolecules, such as monocolonal antibodies which make up the lion’s share of blockbuster pharmaceuticals. However, unlike small molecule or large molecule production, cell therapy products are living, breathing cells, which presents unique bioprocessing constraints and challenges. Greg noted that there are technologies based on monoclonal production that can expand hMSCs in large quantities, like using 3D microcarrier-based bioreactor systems, but there are still many manufacturing innovations required before these manufacturing platforms can support a commercial cell therapy product.  

The technology gaps that he specifically mentioned for upscaled hMSC manufacturing were downstream processing technologies, specifically the unit operations related to:

Enabling a New Paradigm in hMSC Suspension Bioreactor Cultures

Introduction

RoosterBio is introducing a new product for highly efficient bioreactor expansion of human Mesenchymal Stem/Stromal Cells (hMSCs) that we are calling RoosterReplenish-MSC.  This innovative, first-in-class stem cell product is a concentrated bioreactor feed that replaces nutrients and growth factors that have been depleted during microcarrier expansion of hMSCs and replaces the need for a media exchange, enabling scalable and efficient fed-batch hMSC bioreactor expansion processes.  This is the first of several new products that we will launch enabling a cell therapy and tissue engineering bioprocess revolution that will be the foundation of a sustainable Regenerative Medicine Industry.

Media Designed for Scale-up

Human stem cells, characterized by their multi-lineage differentiation potential, tissue regenerative capacity, and high proliferation rates, are the most critical raw material in Regenerative Medicine today. Most cell-based therapies require between 50 million and >1 billion cells per patient application, necessitating efficient expansion (i.e. manufacturing) of starting cell sources.  Today, the most widely used cell expansion platforms for stem cell culture are planar technologies such as flasks and multi-layer cell factories (Rowley et. al), but it is generally accepted that lot sizes and COGS generated from these platforms are insufficient to meet the demand of a widely-used commercial product (Simaria et al).

Production technologies such as single use suspension bioreactors (used routinely in protein, monoclonal antibody and vaccine production) are proven to be robust, scalable manufacturing platforms. These platforms operate in a closed and controlled environment, which minimizes the risk of contamination, and are shown to reduce the time, expense, and carbon footprint required for cell processing. More importantly, it has been shown that such systems can yield lot sizes of hundreds of billions to (eventually) >1 trillion cells per manufacturing run, producing commercially-relevant lot sizes (Rowley et. al). 

MSC expansion in suspension bioreactors is typically done by growing cells on adherent substrates, such as microcarriers (Chen et al, Schnitzler et al, Szczypka et al).  Optimization of an efficient MSC bioreactor culture is central to maximizing yields and recovering healthy, functional cells at harvest. Another key attribute to efficient manufacturing processes is cost, and minimizing cost is crucial for building successful business models around MSC-based regenerative therapies.  Cell culture media is consistently the main cost driver of any stem cell production process, and it is critical to minimize media usage to keep production costs to a minimum (Rowley et al). Optimization of MSC-microcarrier cultures typically involves either full or partial media exchanges to manage nutrient supply and waste build-up (Goh et. al, Reichmann et al, Nienow et al, Santos et al, and Heathman et al), which is expensive and impractical at larger scales of >50L culture. This media exchange mentality is driven by the fact that commercially-available hMSC media formulations have been designed for flask-based culture processes and full media exchanges.  

Half media exchanges are the simplest to perform in small scale; however, when spent medium is only partially replaced with growth medium, the final concentration of nutrients and growth factors required for optimum cell proliferation are significantly reduced, resulting in lower cell proliferation rates. In addition, this procedure is time consuming, and the feasibility at larger scales decreases. Fed-batch culture, on the other hand, is more efficient in reducing processing time, mitigating contamination risk, and reducing costs associated with waste management such as time, labor, equipment and facility required to prepare and handle spent media. Hence, a new media design philosophy is required for suspension-based hMSC culture, and RoosterReplenish-MSC, coupled with RoosterBio’s High Perfromance Media kit, is the first media system designed specifically for hMSC bioreactor culture.

RoosterReplenish-MSC, a concentrated bioreactor feed, replaces nutrients and growth factors that have been depleted from RoosterBio’s High Performance Growth Media (KT-001) during extended culture. The nutrient boost provided by RoosterReplenish-MSC replaces the need for partial or full media exchanges when using our rich basal media, yielding a more streamlined culture process for hMSC expansion in bioreactors, and enabling efficiency in media utilization. 

In the next section, we will describe a series of studies performed with RoosterReplenish-MSC in microcarrier suspension culture.

Experimental Methods, Results & Discusssions

How Quickly are Cell-based Products Really Developing? Thoughts from the IBC Cell Therapy Bioprocessing Conference

Last week was the 4^th Annual IBC Cell Therapy Bioprocessing Conference.  IBC (home of the BioProcess International conference) was the first conference organizer to dedicate a focused meeting on Cell Therapy Manufacturing Technologies 4 years ago.  Since the first conference in 2011, the growth in the field, and the conference, has been amazing.  The attendance has grown from less than 90 in year 1 to over 200 this year.  The content has also evolved heavily over the last 4 years, demonstrating a high level of sophistication and maturity in a field that seems “early stage” to those looking in from the outside.  The talks this year increased in the amount and quality of data presented. Topics included the impact of automation on the simplification, streamlining, and cost reduction of autologous therapies, the use of Quality by Design (QbD) in bioreactor scale-up and analytical development, advances in tissue engineering and biofabrication techniques, and even 2 year data on marketed products.   Phil Vanek, the General Manager of GE Healthcare’s Cell Therapy business, summed it up during his talk where he stated that: GE is interested in 1) big problems, 2) compelling clinical data, and 3) opportunities for “industrialization”, and “Cell Therapy/Regenerative Medicine has all three”.

Various cell manufacturing and processing devices seen throughout the exhibits at IBC's
4th Annual Cell Therapy BioProcessing Conference - No BioPrinters (yet!)

There are many signs that the Cell Therapy field is moving much faster than the protein therapeutics field before it and demonstrating rapid progress.  What we have here is a traditional case of  “standing on the shoulders of giants”, which has been paraphrased on Wikipedia as "discovering truth by building on previous discoveries”.  

Scale-up Production of hMSCs: Highlights from the BioProcess Summit Cell Therapy BioProduction Sessions – Post 2 of 2

From www.CellTherapyWonk.com

Fall is almost here, and that means it is Cell Therapy BioProcessing and Manufacturing conference season.  This year it started a few weeks earlier as conference organizer CHI put together a Cell Therapy BioProduction session as part of their Annual BioProcessing Summit in Boston from August 18-22.  In our last post (Scale-up Production – Post 1 of 2), I summarized some of the cool technologies that vendors had on display, as well as some of the poster highlights.  In this post, I want to highlight just a few talks that were focused on manufacturing, scale-up and Cost of Goods of allogeneic cell therapies.  There were several other great talks, but I just wanted to focus on these three due to topic and brevity.

Manufacturing, Cost of Goods, and Unprecedented Stem Cell Process Yields:

On the first day, we had a dynamic duo from Loughborough University give a pair of excellent talks.  Experienced Manufacturing Engineer David Williams gave a great talk on precision manufacturing of living products, highlighting the challenges of working with the inherent variability that comes with primary cell culture.  Dr Williams is the Director of the Center for Innovative Manufacturing in Regenerative Medicine that “works to equip the regenerative medicine industry with manufacturing tools, technologies and platforms by considering the ‘right therapy, right patient, right time’ supply chain from end to end.”  In his talk, he highlighted the need for solid quality characteristics so you know exactly what you are manufacturing – and can do it “again, and again, and again, and again….”.  He points out that, without knowing what characteristics are important for your product (the identity and functional potency of your cells), you: can’t manufacture to specification, can’t scale up, can’t implement new raw materials in your process, can’t transfer manufacturing to another facility, and can’t reduce COGS through process optimization.  Hearing David talk is always a reminder of how important the basics are.

Scale-up Production of hMSCs: Highlights from the BioProcess Summit Cell Therapy BioProduction Sessions – Post 1 of 2

Closed system 10 layer and media bags are 

now readilyavailable from several vendors.

Fall is almost here, and that means it is Cell Therapy BioProcessing and Manufacturing conference season.  This year, it started a few weeks earlier as conference organizer CHI put together a Cell Therapy BioProduction session as part of their Annual BioProcessing Summit in Boston from August 18-22.  It is always great to see new conferences including Cell Therapy content, as it shows the maturation of the field.  There is a now a “market” that the organizers believe is worth creating content for.

CHI was kind enough to invite RoosterBio to give the kick-off presentation, so I was able to get re-immersed on all that is new and improved in Cell Therapy Manufacturing Scale-up.  This blog post is meant to share a few of the interesting observations from the meeting and will focus on highlights from the vendor exhibits (i.e. product innovations) and the posters.  The next blog post will share some take-homes from the talks.  There were indeed some valuable and exciting new reports that I want to communicate. 

One key observation is that, while it is still my belief that most of the Allo-products are currently manufactured in 10-layer culture vessels (see above pic), most of the presentations focused on the next generation bioreactor-based processes.  There will continue to be a major shift over the next few years to more automated platforms such as these.

Products on Display for BioProduction of Therapeutic Cells

I always find it worth noting what products the vendors have on display, as they will be developing products based on requests from the market – so more new products displayed means "growing market", which turns into better tools available for everyone.  Interestingly, even at a general BioProcessing conference, there were several booths with Cell Therapy-focused products, and there were 3-5 posters (out of maybe 30) on the scale-up and processing of human MSCs (hMSCs). 

There were a few product innovations and focused product areas among the vendors that I want to highlight here (and we are not getting paid for this, I promise – no sponsorship at all).

Ready-to-Use Microcarriers from Pall

·         Pre-sterilized and ready-to-use microcarriers.  Both Pall/Solohill (see pic) and Corning now offer these products in bottles, but more importantly, in closed system bags ready to seed into a bioreactor system.  Microcarriers in the past had to be prepared and autoclaved by the end user, creating work and yet another set of variables to control when trying to implement this technology.  By providing pre-sterilized microcarriers that are QC’d for efficient cell attachment (I am assuming this QC step; I will try to confirm that), this takes one less process step out of the hands of the process development scientist and makes implementation that much simpler.  This is an important advancement in the field.

·         PBS Biotech reported hMSC densities (expanded in their bioreactors) consistently north of 1 million cells/mL, and up to 3 million cells/mL in a serum containing media.  These numbers are a good 10-fold greater than any number published or presented just 5 years ago, and the highest and most consistent I have seen presented to date.  It is unclear if the reason they achieved these targets was due to the microcarrier and media combination, their novel low shear bioreactor design, or just plain good bioprocess engineering - likely a combination of all three.  In any case, it is a significant achievement, and it demonstrates that it can be done.  Other vendors will now be trying to beat it, and cell therapy companies will be trying to implement it.

·         Vendors are beginning to focus, at least some, on the post-expansion/downstream processing (microcarrier removal, cell concentration) of the cells as well.  Millipore had a presentation and a poster that discussed microcarrier removal using single use filters and the concentration of hMSCs post-harvest using scalable tangential flow filtration (TFF) technology – both with good post-processing viability and recovery.  See poster summary below.  Downstream processing continues to be an under-appreciated aspect of the field and cannot be an afterthought to scale-up culture.  If you scale your expansion to several hundred liters before beginning to think about how you will process the massive cell volumes you have, it could set your program back over a year while these technologies are developed and integrated into the manufacturing process.  It is good to see these aspects of manufacturing get some focus here.

Poster Highlights: