Building Effective Multi-Year Process Development Programs: Evolution of Technology Platform Decisions Based on Lot Size

Authored by Josephine Lembong, Ph.D., Scientist, Product & Process Development & Jon Rowley, Ph.D., Founder and Chief Product Officer, RoosterBio Inc.

Previously, we published a blog post on estimating hMSC lot sizes for clinical manufacturing, with the goal of outlining a development program that could tailor accordingly. This exercise is crucial because the calculated target cell lot size dictates the final production platform needed for your therapeutic product. The next step would be to determine the appropriate manufacturing platform, for each unit operation, that will meet the calculated hMSC lot sizes throughout clinical development. Having a solid, multi-year plan will help your company succeed at navigating this complex maze that is the path to market success.

“It is the technologist’s and engineer’s jobs to drive the technology platform decision making process”

The final decision of production platforms can be overwhelming; even though there is a certain goal in mind for the present time, you do want to keep it flexible and scalable for other potential applications in the future. There is also the goal of managing through the “Comparability Challenges” as these changes are implemented. Adding to the complexity is that as the RegenMed industry grows, the technology providers of cell processing platforms across the various unit operations seems to be increasing in a fractal nature, with little standardization across devices. These technologies (e.g. bioreactors, continuous centrifuges, fill finish/controlled freezing, and other automation platforms) are significant investments to the company in the form of cost and time. It is the technologist’s and engineer’s jobs to drive the technology platform decision making process by derisking these technologies and establishing a multi-year development program, all while determining the costs associated with the program, and communicating these needs to the company’s business team so that they can raise the needed capital for these programs over time.

The goal of this post is to lay out the various scales of hMSC production and highlight the existing technology platforms for the different unit operations involved in the manufacturing process. This will help define the requirements that will guide the company’s multi-year process development program to meet projected future lot sizes.

Each phase of a clinical trial is associated with a specific production scale, which dictates the production platform

At the end of the last post, we arrived at the following estimated lot sizes based on a set of assumptions: 525 billion viable hMSCs per final commercial manufacturing lot, assuming a mid-range dose for a cardiac indication aimed to treat 100,000 patients per year, with a relatively safe, conservative assumptions regarding losses in cell viability and recovery during every step of the production process. Assuming a go-to-market lot size of 20% of the full commercial scale, we estimated that one could target a 100B cell lot size for Phase III, a 25B cell lot size for Phase II, and potentially a 10B cell lot size for a Phase I trial. These are simply guidelines that will change based on assumptions, but we recommend everyone go through this exercise (as outlined here) for each therapeutic program.

Building Effective Multi-Year Process Development Programs: Estimating hMSC Lot Size Ranges for Clinical Manufacturing through Commercial Demand – it is all about the assumptions

Authored by Josephine Lembong, Ph.D., Scientist, Product & Process Development & Jon A. Rowley, Ph.D., Founder and Chief Product Officer, RoosterBio Inc.

Jon Rowley, RoosterBio’s Founder and Chief Product Officer, gave a talk at ISCT 2018 in May and had a set of slides about estimating lot size that had a lot of people scrambling to take notes. We thought we would share the content here at the RoosterBio blog with more discussion and make the content broadly available and open for discussion.  

The concept is consistent with all good strategic planning; 

1. Understand what the future looks like and work backwards,
2. Create a model using reasonable to conservative assumptions along the way to estimate a range of needs,
3. Use this model to lay out what the next several years will look like assuming successful achievement of intermediate milestones, and create multi-year programs that are right-sized, with the right technology platform, for the stage of product / clinical development of your company.

This is the type of consultation that we provide in our Process Design and Acceleration business unit at RoosterBio, which we offer to our customers that are incorporating RoosterBio hMSC cell banks and bioprocess media systems into their product and process development efforts. However, we love to share our knowledge with the broader RegenMed Industry and are offering it up here.

Notes to readers: this exercise has the base assumption that this is an Allogenic, Universal Donor manufacturing process [1] and is not applicable to Autologous or patient-specific products. The core logic would still hold when applied to autologous, but at a much smaller scale.

Lot Size Targets Dictate the Production Platform 

The goal of any process development program is to create a right-sized manufacturing process for your immediate business goals, but with future scalability in mind. Understanding the future lot size requirements will help strategically align manufacturing requirements today with commercial scalability while laying a platform foundation to minimize comparability risks.

First Start with the End in Mind – Engage with Marketing

Get with the business team within your company (usually Strategic Marketing and/or Commercial Operations) and request a numbers-driven discussion on which of the multiple indications that a process development program should be built for. There are usually several therapeutic indications of interest, but it is important to realize that a manufacturing process for an ocular indication that has a dose of 1 million cells/dose is very different than a Crohn's disease indication that has a dose size of greater than 1 billion cells/dose [2]. Each therapeutic indication from a company must be treated as a distinct product and target, with a distinct process. Just because the same cell type is used in both therapeutic products does not mean they share a manufacturing process. This is critical to bring clarity to each program.

“A common pitfall that many cell therapy companies fall into is that each therapeutic indication is not addressed as a unique product with a unique manufacturing process”

Once the team is aligned on the target indication (or set of prioritized target indications), then you need to understand what a reasonable peak commercial market demand would be – this is likely to be found in the business model projections that are built for investors and is a good place to start. If there are 500,000 patients a year in your target indication and the business is aiming to capture 20% of them (or treat 100,000 patients/year) at peak commercial demand, then you need a process that will scale with that over time, assuming both clinical and market success. It is a good idea to establish a range. We often like to perform this exercise with a low, middle and high assumptions (such as 30,000 patients as low, 100,000 as the target, and 250,000 patients treated/year if wildly successful). For this article, we will simply focus on the '100,000 patients treated assumption' for our calculations.

Dosing Assumptions: this is one of the hardest parts

Estimating dosing is sometimes the most difficult part. Does the patient need a single dose or multiple doses, and how many cells per dose? In many cases a dose escalation trial has not yet been performed, so some educated guesstimates are required. Taking a low/mid/high range of estimates also works here, and there is sufficient published work related to hMSCs in different indications that you can get close enough for these purposes [2,3]. For this example, we will assume a cardiac indication. There is a good amount of published work on hMSCs for cardiac indication and we can assume a conservative low dose of 25 million cells, a mid-range dose of 50 million, and a high dose of 100 million [4,5]. In this blog post we will perform all calculations with an assumption of 50 million cells per dose.

Calculating Yearly Product Needs at Peak Commercial Demand

• At peak demand, we aim to produce 100,000 doses per year, with a size of 50 million cells per dose.
• 100,000 doses per year ≈ 2,000 doses per week
• Weekly production is not recommended – it is simply unsustainable, so let’s assume one (1) lot production every two (2) weeks, or 25 lots total in a year (which is still a lot for any manufacturing site)
• Not all lots will be successful – assume a 10% scrap rate, thus 2-3 lots are lost per year, giving us 22-23 lots per year
• 100,000 doses per year / ~23 lots per year = 4,450 doses/lot 
• Assume 10% of lot goes to testing (~450 dose), so a final target lot size of 4,900 doses/lot will be needed to be manufactured at peak commercial demand

It is important to remember that it typically takes multiple years for a successful therapeutic to reach peak demand, so it is not necessary to go to market with a manufacturing process capable of meeting peak demand – especially for an early stage field like Regenerative Medicine. We will create estimates for peak demand, but then focus on building a reasonable “go-to-market” lot size and manufacturing process.

Calculating Total Cells to Manufacture that are Required to Fill into the Final Container

• We will assume 1 dose goes into one container.
• We assumed a mid-range dose size of 50 million (i.e. 50 million viable cells per vial, post-thaw).
• Cell Count is a strict QC parameter with a straight forward specification. It is important to point out that often there is a solid safety factor, or overfill, applied to this metric. You never want to fail a lot on cell count after you spent hundreds of thousands of dollars creating the units – so the risk averse will overfill vials until sampling and cell enumeration is an accurate, consistent, and precise method.
• Assume 15% (worst case) viability drop and 20% recovery loss during cryopreservation (these assumptions should be data driven from your process, and constantly trended and updated during manufacturing).
• Based on the above assumptions, to consistently achieve 50 million viable cells after thaw, it is required to target filling at 75 million viable cells into the final container:
• 75 million viable cells – 15 million (20% total cell loss on recovery) = 60 million viable cells 
• 60 million – 9 million (15% viability drop) = 51 million viable cell target specification
• NOTE: some programs will only overfill by 10-20%, but we find that with this range it is very difficult to routinely obtain successful cell counts in a manufacturing/QC environment, so we recommend 30-50% overfill for calculations.

Calculating Lot Size Requirements at Peak Commercial Demand

One unfortunate fact of cell manufacturing is that you need to manufacture more cells than you need to fill, since there will be losses associated with the post-harvest processing steps. Many biological manufacturing processes have losses in the 40%-60% range in this “downstream” processing.  For our process, we go through the following math:

• 75 million cells/dose × 4900 doses per manufacturing lot = 368 billion viable cells needed to target 4900 doses from every manufacturing lot.
• We assume 30% post-harvest cell loss during processing for this exercise – thus at least 525 billion cells need to be manufactured in order to have 368 billion cells during the fill unit operation.
• 525 billion viable cells – 157 billion (30% post-harvest cell loss) = 368 billion viable cells 
• 30% cell loss does seem significant, however every process development group will have a team working to optimize their process to minimize this number.  It is likely that recoveries from earlier processes will be much greater than 30%, and with optimization at large scale it is possible to improve.  For modeling, it is always worth using reasonable to conservative estimates.

In conclusion, we arrived at the following numbers:

• At peak commercial demand, 23 successful lots of 525 billion cells per lot need to be manufactured in order to meet the demand, assuming a mid-range dose for a cardiac indication aimed to treat 100,000 patients per year. 

This calculation does not take into account further downstream losses associated with stability, multiple distribution sites, safety stocks – which will just make the numbers worse. For this exercise, we will stop here. The 525 billion viable cells per lot is the number we were looking for, and it is this number that will drive the production platform decisions for streamlined hMSC clinical manufacturing.

For an early-stage RegenMed company, a go-to-market lot size of ~20% of the “peak commercial scale” is a reasonable target to plan for, so a ~100 billion cells/lot would be a good target number to develop and run your Phase III trial at.

We’d like to summarize this post with a few number recommendations:

Phase III/Go-to-Market: ~100B cells

Phase II (100-200 patients over 3-4 lots): 15-25B cells – this is an intermediate step up from Phase I and within a log of the Phase III/go-to-market process.

Phase I (small scale): 5-10B cells

The next stage is to lay out the technology platforms for the various manufacturing unit operations that are capable of processing these cell numbers. This will be the topic of our next blog post.

References:

1. Simaria et al., 2013. Allogeneic cell therapy bioprocess economics and optimization: Single‐use cell expansion technologies. Biotechnol Bioeng 111(1): 69-83. doi: 10.1002/bit.25008 
2. Olsen et al., 2018. Peak MSC—Are We There Yet? Front Med 5:178. doi: 10.3389/fmed.2018.00178 
3. Squillaro et al., 2016. Clinical Trials With Mesenchymal Stem Cells: An Update. Cell Transplant 25(5):829-848. doi: 10.3727/096368915X689622 
4. Majka et al., 2017. Concise Review: Mesenchymal Stem Cells in Cardiovascular Regeneration: Emerging Research Directions and Clinical Applications. Stem Cells Transl Med 6(10):1859-1867. doi: 10.1002/sctm.16-0484 
5. Golpanian et al., 2016. Concise Review: Review and Perspective of Cell Dosage and Routes of Administration From Preclinical and Clinical Studies of Stem Cell Therapy for Heart Disease. Stem Cells Transl Med 5(2):186-191. doi: 10.5966/sctm.2015-0101

ISCT 2018: MSC Biomanufacturing, Bioprocessing, Scale-Up, Analytics and Exosome Production Take Center Stage

Authored by Katrina Adlerz, Ph.D. Scientist, Analytical Development, RoosterBio Inc.

RoosterBio attended the International Society for Cell Therapy annual meeting, held May 2-5, 2018 in Montreal, which brought together leaders in the field including academic researchers, industry scientists, regulators, and clinicians. The society and conference focus on three key areas of translation: Academia, Regulatory, and Commercialization.

Six Roosters attended to hear the latest research and translational innovations at the scientific talks, present three different posters, and man the booth in the exhibit hall. Jon Rowley, founder and CTO of RoosterBio, also gave two talks discussing innovations to accelerate MSC Biomanufacturing, Bioprocessing and Scale-Up. He shared insights from RoosterBio’s path to GMP-manufactured cells and media as well as strategies for overcoming obstacles in his talk “Technologies for Radically Reducing Development Timelines of hMSC-based Therapeutic Products” during the Strategies for Commercialization Session. (New to MSCs? Read more about MSCs and biomanufacturing here, here, here and here.) For a copy of Jon's talk, email us.

RoosterBio Analytical, Process & Product Development efforts were well-represented with three posters.

- “A Xeno-Free Fed-Batch Microcarrier Suspension Bioreactor System for the Scalable and Economic Expansion of hBM-MSCs” showed that MSC critical quality attributes were maintained in 0.1L and 3L bioreactor culture. Poster.

- “Scalable Xeno-Free Manufacturing of Extracellular Vesicles Derived from Human Mesenchymal/Stromal Stem Cells” explained a process for generating a high yield of EVs/Exosomes from MSCs in a shortened time frame using RoosterNourish^TM-MSC-XF Media. Poster.

- “Development & Technology Transfer of a cGMP Potency Assay: Testing of an Ancillary Material for Stem Cell Manufacturing” outlined the steps in assay development, assay qualification/validation, and tech transfer for a custom potency assay based on cell expansion. Poster.

- In addition, the RoosterBio , BioLife Solutions and Brooks Life Science teams collaboratively presented a poster on MSC cryopreservation: "The Effect of Cryomedia Selection and Transient Warming Events on Post-Cryopreservation Human MSC Function". Poster. 

The posters gave presenting scientists the opportunity to talk with those doing similar work in process or assay development. It allowed us to learn from and share our expertise with the community.

The conference kicked off with a RoosterBio-sponsored workshop: Improving Mesenchymal Stem Cell Potency and Survival. Steven Bauer, Branch Chief of the Cellular and Tissue Therapy Branch  of the US FDA, and Head of the FDA's MSC Consortium, discussed his innovative work developing predictive assays for MSC potency by analyzing cell morphology in his talk “High Throughput Approaches to Assess MSC Function”.  You can find his blog here.  There were also a number of talks discussing clinical trials results. A common theme of the session was the need to develop analytical methods and assays that can predict MSC-based treatment efficacy in patients.

Good Manufacturing Practice for Cell and Cell-Based Therapies: Facilities & Quality Control

Novel cell and cell-based therapies require stringent manufacturing, testing and oversight to ensure integrity, function, and above all else, patient safety upon administration.  Tissue-derived cellular products are considered to be manufactured products and are regulated as such.  Thus, you must ensure that your cell manufacturing process is aligned to current Good Manufacturing Practice requirements.  (Note the “current”.  This means that these are evolving requirements, so you must stay up-to-date!)  In the United States, human cells, tissue and cellular- and tissue-based products (HCT/Ps) are regulated by the Center for Biologics Evaluation and Research (CBER), a division of the U.S. Food and Drug Administration (FDA).

When manufacturing cell and cell-based products, a production facility under strict Quality Control must be used.  Ideally, this facility includes the cell manufacturing suites, the storage space for raw and finished product and any laboratory/testing areas.  Thus, (1) facility design, access and maintenance, (2) equipment purchase, installation and operational qualification (I/OQ), use and maintenance and (3) raw material specifications, purchase, use and storage must all be carefully controlled.  For cell and cell-based therapies, terminal sterilization of the final product is often not possible.  As such, quality by design (QbD) is highly important in cell therapy, with stringent testing conducted on the tissue Donor (our next blog post in this series will cover this topic) to preclude risk of contamination at the source.  In addition, facility and equipment standards and monitoring must be instituted to ensure aseptic processing of cell and cell-based products.  To this end, closed systems and single-use disposables should be used whenever possible to minimize risk of contamination.  Therefore, a cGMP manufacturing facility must include clean rooms which control for temperature, humidity, pressure and air particulates, preventing any contamination of manufactured product due to the environment, materials, human handlers and cross-contamination from other products manufactured in the same facility.  As such, there should be uni-directional flow of materials and people through these areas and personnel must follow proper gowning procedures.  Facilities and equipment requirements are defined under US FDA 21CFR§211 and 21CFR§1271.

As mentioned above, stringent Quality Control systems must be in place to qualify all reagents and processes and to institute Standard Operating Procedures (SOPs) to ensure quality and consistency in manufacturing processes and the end product. 

Facility and Quality Control considerations for cGMP cell manufacturing.

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:

Comparability of hMSC Economic and Quality Attributes after Expansion in Bovine Serum Containing vs Xeno-Free Bioprocessing Media Formulations

Human Mesenchymal Stem/Stromal Cells (hMSCs), from bone marrow or other tissues, are poised to have the most significant impact on Regenerative Medicine compared to any other single cell type.  This is due to their ability to be utilized across multiple therapeutic indications due to the wide ranging functional nature of the cells (1-3).  hMSCs are not only capable of differentiating into tissue-specific cell types, but also have angiogenic, immunomodulatory, anti-inflammatory and anti-bacterial abilities (4).  hMSCs are true Tissue Repair Cells – setting the stage for all phases of wound healing and tissue repair: promoting new blood vessel growth, reducing inflammation to aid healing, secreting several mitogenic factors important for tissue building and stimulating tissue-specific stem cells. 

However, hMSCs have traditionally been challenging to source in significant volumes and at sufficient quality levels, hindering the advancement of the science into medical products.  At RoosterBio, we focus on transitioning hMSCs from a scarce into an abundant resource, and we achieve this by borrowing best practices from the Manufacturing Sciences and applying them towards the grand challenge of producing billions of hMSCs, with critical quality and functional parameters in place, and at costs and volumes that enable the rapid and wide-spread adoption of hMSC technology into clinical practice.

RoosterBio came to market 2 years ago with hMSC cell and media systems that include a highly efficient hMSC bioprocess expansion media  that simply and consistently produces greater than 100x expansion of cells with 8-10 days of culture. Our cell and media system was designed for a “batch” culture process (no media exchange required between passages), removing labor-intensive and costly media exchanges, and enabling rapid expansion with little in process intervention (thus fewer risks for contamination).  While the cell and media system has now been used in several translational and high impact publications (5-8), the expansion medium does utilize low levels of high quality bovine serum to maximize the performance and robustness of the overall system. 

In recent years, the field has been shifting towards xeno-free (XF) cell and media systems to remove any remaining safety issues related to xeno-sourced animal components (9-13). Furthermore, our customers have been requesting XF expansion options. We have listened to our customers and spent the last year developing and optimizing a fully XF media formulation based on our innovative bioprocess media platform.  The goals of this media were to remove all xeno-sourced raw materials from the formulation, while maintaining all hMSC functional properties, as well as the economic and production efficiency of our initial bovine serum containing (BSC) media formulation.  We are now ready to commercially launch our XF media to advance the industry, and this blog post will outline the initial work we have performed to evaluate the comparability of expansion, cost and functional properties of hMSCs expanded in the new XF media compared to our flagship BSC media.

Table 1. Media formulations and nomenclature.

For the purpose of this blog post, we will be comparing RoosterBio hMSC products expanded in either our initial bovine serum containing High Performance Media, or our new xeno-free High Performance Media XF formulation.

METHODS

Cell expansion. RoosterBio hBM-MSC were expanded in BSC Media and XF Media. Frozen cells were thawed and plated in triplicate at 3,000 cells/cm² in T-75 flasks and cultured for 4 days. At 4 days, cells were harvested with TrypLE (Gibco) and cell number and viability were determined on a Nucleocounter. These cells were used for the analyses below or plated again for further expansion.

Cell surface marker expression. To determine if the cells grown in XF Media were capable of expressing MSC markers, hBM-MSC expanded in both BSC and XF Media were plated and incubated in DMEM/10% FBS for 5 days prior to flow cytometry.

Immunomodulatory function. Induction of indoleamine 2,3-dioxygenase (IDO) activity by exposure of hMSCs to the pro-inflamatory cytokine IFN-γ is central to the immunosuppressive function of hMSCs (14,15). See here for a blog post on this topic. hBM-MSCs were expanded in BSC and XF Media (Donors 1 and 2) or XF Media alone (Donor 3), harvested and plated in High Performance Basal medium (SU-005) with 2% FBS at 40,000 cells/cm². After 18-22 hr of incubation, cells were treated with IFN-γ (10 ng/ml) for 24hr±1hr. The cell supernatant was collected, and the kynurenine concentration was measured using a spectrophotometric assay and normalized to number of cells and days of incubation to obtain the amount of IDO secreted (expressed as pg kynurenine secreted per cell per day).  

Angiogenic cytokine secretion. hBM-MSCs were expanded in BSC or XF Media, harvested and plated in High Performance Basal Medium with 2% FBS at 40,000 cells/cm². After 24hr±1hr culture supernatant was collected and assayed for FGF, HGF, IL-8, TIMP-1, TIMP-2 and VEGF concentration using a MultiPlex ELISA (Quansys). Cytokine concentration was normalized to number of cells and days of incubation to obtain cytokine secretion rates.

Trilineage differentiation. hBM-MSCs were expanded in BSC or XF Media, harvested and plated in High Performance Basal Medium with 2% FBS at 5,000-10,000 cells/cm²  for adipogenesis and osteogenesis or formed into 100,000 cell micromasses for chondrogenesis. On day 1, cells were switched to differentiation or control media (LifeTech StemPro Differentiation Kits) and cultured per kit protocols for 10-21 days. Differentiation was detected by Oil Red O (adipogenesis), Alizarin Red (osteogenesis), or Toluidine Blue (chondrogenesis) stains.

COMPARATIVE ANALYSES

Cell expansion. A key characteristic of RoosterBio hMSC cell and media systems is rapid cell expansion with a guaranteed 10-fold expansion within 7 days. In engineering our XF Media system, we aimed to preserve this hMSC expansion profile.  hBM-MSCs display rapid and comparable growth in both our BSC Media and the new XF Media formulations, with similar doubling times and expansion rates.  We see the typical variability across donors, but all donors are harvested at greater than 30,000 cells/cm², after plating at 3,000 cells/cm², within 5 days (Figure 1). hBM-MSC growth over 2 passages yields greater than 1 billion cells using both our BSC and XF Media (and 10M cell product vials) in less than 2 weeks (Figure 2), leading to tremendous economic benefits (described below).

Figure 1. hBM-MSCs expand efficiently in XF Media. All cell lots expanded at least 10-fold (>30,000 cells/cm2) in XF and BSC Media (data shown for 2 donor lots). Data are mean of 3 replicates +/- SD.

Towards a Cell Therapy Manufacturing Technology Roadmap -- Resources

RoosterBio was founded to accelerate the development of the Regenerative Medicine field by utilizing advancements in Manufacturing Sciences.  As such, we have long been involved in laying the groundwork for a Technology Roadmap for sustainable Cell Therapy Manufacturing.  

Recently, RoosterBio was part of the first-ever Cell Manufacturing Consortium aiming to position the United States as a leading developer of Cell Manufacturing Technologies and the Chief Authority on Cell Manufacturing Standards Worldwide.  The goal of this Consortium was to establish a collaborative public-private partnership that engages industry, academia, regulators, and other stakeholders in removing barriers to the advancement of the cell-manufacturing industry, thereby bringing new therapies and diagnostics to the healthcare market.  As a result, the Consortium members have formulated an extensive Cell Therapy Manufacturing Technology Roadmap to the year 2025.  This document is in final review and should be available shortly.  In the meantime, we've compiled some resources (to which we will continue to add and seek your input as well) on Cell Therapy Manufacturing for those interested.

Relevant Publications:

• Developing Cell Therapy Biomanufacturing Processes
• Cell Therapy Bioprocessing
• Meeting Lot-Size Challenges of Manufacturing Adherent Cells for Therapy
• Developing assays to address identity, potency, purity and safety: cell characterization in cell therapy process development.

The Use of Animal Serum in the Clinical Translation of hMSCs

Human mesenchymal stem or stromal cells (hMSCs) are an integral part of cell-based therapeutics, with over 400 clinical trials recently completed or in progress using hMSCs. As more research teams transition their stem cell-based regenerative technologies to the clinic, the use of serum in the cell production process has been, and will continue to be, a necessary evil that must be managed.  Luckily, pharmaceutical regulatory agencies, driven by the biologics industry over the last 30 years, have established guidances and guidelines that have helped to demystify and clarify some critical aspects of dealing with animal components. As it is important to have an understanding of how to manage serum during the clinical translation of hMSCs, we have focused this blog post on this specific topic

There are many researchers in the MSC community who firmly believe that the FDA simply does not allow hMSCs into clinical trials if the cells have been cultured in media supplemented with animal serum. This is currently not the case, and in fact, Mendicino et al. from the Center for Biologics Evaluation and Research at the FDA reviewed all MSC regulatory filings and found that over 80% of all regulatory submissions described the use of fetal bovine serum (FBS) during the hMSC manufacturing process [1]. Several other analyses of hMSC-based clinical trials in recent years have similarly shown that at least 65-75% of trials utilize FBS [2,3,4]. Regardless, a push to remove serum from the manufacturing process still continues due to regulatory, production and supply chain concerns.  Each of these areas is detailed below.

(Note: when we refer to "clinical-grade" products below, this is not an official regulatory classification, it is meant to generally refer to materials that are destined for use in clinical testing of cell therapies.)

Regulatory Compliance      

The main regulatory concerns associated with the use of xenogeneic serum include the risk of contamination with non-human pathogens and inducing an unwanted immune response. To account for such serious consequences, the FDA has put several requirements in place for the production process of both clinical-grade FBS and hMSCs:

• FBS: Clinical-grade FBS must be derived from cattle herds grown in countries that are USDA approved for import, with well-monitored animal health status [2]. The FBS should be processed under current good manufacturing practice (cGMP) standards that set minimum requirements for the facilities, materials and protocols used [2]. Every batch or lot of FBS must be traceable back to its country, slaughterhouse and herd of origin. Finally, all lots must be tested for adventitious agents (viral contamination), sterility (bacterial and fungal elements), endotoxin levels, mycoplasma content and other constituents [2,5]. While regulatory agencies address safety, it is up to the cell manufacturer to establish metrics around performance, as FBS has traditionally been both a major cost driver and a source of process variablity.
• Clinical-grade hMSCs: Clinical-grade hMSCs must be manufactured under cGMP standards, and this topic is covered extensively in the literature.  As it pertains to serum use, each lot of serum used during the cell production process must be documented [5], and the final cell product must meet specific standards of identity, potency, purity and safety. Purity standards include freedom from unwanted contaminants (such as other cell types, endotoxins, residual proteins and animal serum) [6]. The FDA Code of Regulations for Biologics provides a guideline for vaccines that animal serum levels must be under 1 ppm in the final product formulation when serum is used in any part of the process [US FDA. 21 CFR 610.15 ].  While there is no direct guidance for cellular therapies, the 1ppm residual level has been used as a target in some cell therapy manufacturing processes [6] and is a good place to start when developing process specifications.

These checks and balances have allowed clinical trials using FBS-cultured hMSCs to be conducted safely. A meta-analysis by Lalu et al. showed that there was no evidence of infection or toxicity in any subjects involved in clinical trials using FBS-cultured hMSCs [7]. Several other clinical trials have described the use of FBS in cellular therapeutics and biologics without any adverse side effects [8-12]. That said, it is best practice to develop sufficient cell washing protocols after cell harvest, and before formulation, to remove process impurities and get serum protein levels down to acceptable levels [13].

For FDA resources on this topic, see:

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

Cryopreserved hMSCs maintain comparable in vitro functional activity compared to fresh hMSCs

INTRODUCTION:

Human mesenchymal stem cells (hMSC) are currently in use in over 400 clinical trials and are critical components of tomorrow’s cell-based products and devices (1, 2, 3). Secretion of biomolecules by hMSC influences many biological processes and is thought to be central to the mechanism of action. Since widespread clinical use of hMSC and cell-based therapies with positive economic outcomes will be facilitated by frozen storage, cryopreserved hMSC must maintain high levels of biological function upon thaw.  Additionally, while hMSC have an excellent clinical track record in terms of safety, efficacy data has been difficult to come by, suggesting that more standardized cell formats are needed.  This too could be addressed by effective means of cryopreservation, allowing off-the-shelf hMSC products to be widely used in Regenerative Medicine, Tissue Engineering and for 3D BioPrinting of cells and tissues.

To date, there have been conflicting results on the impact of cryopreservation on hMSC function.  The Galipeau lab showed that cryopreserved MSC have impaired immunosuppressive function in response to the pro-inflammatory cytokine, IFN-γ (lower IDO response, and decreased T-cell suppression) relative to proliferating cells (5, 6). The LeBlanc group similarly found that cryopreserved hMSC have reduced responsiveness to IFN-γ, decreased production of anti-inflammatory mediators, and impaired blood regulatory properties (7). In contrast, other studies support the use of cryopreserved hMSC.  The Mueller lab showed that cryopreservation of hMSC did not change the cells’ immunomodulatory activity, viability, or differentiation (8). The Weiss group also performed in vivo tests of thawed hMSC and found that “in an immunocompetent mouse model of allergic airways inflammation … thawed MSCs are as effective as fresh MSCs.” (9)  The difference in results is likely due to differences in the cryopreservation formulations, controlled rate freezing protocols, and how the cells are thawed and handled prior to implantation.

To address the critical issue of cryopreservation in our hMSC systems, we compared the biological activity of RoosterBio hMSCs from 2 donors either (a) with cells straight out of cryopreservation (THAW) or (b) with cells that had been in culture for at least 5 days (FRESH), while controlling for PDL.  Based on the literature, we established a conservative  hypothesis for this study that cryopreserved hBM-MSC would exhibit diminished immunosuppression and altered angiogenic cytokine secretion compared to proliferating hBM-MSC in response to challenge by inflammatory cytokines. We tested this hypothesis with RoosterBio’s hBM-MSC, produced with GMP-compatible and scalable manufacturing processes, by comparing the immunomodulatory activity and angiogenic cytokine secretion of proliferating (FRESH) to cryopreserved and thawed (THAW) hBM-MSC.  By presenting the results of this study, we hope to provide additional data points for the industry on the use of cryopreserved, off-the-shelf hMSCs for Regenerative Medicine, Tissue Engineering and 3D BioPrinting.

METHODS AND EXPERIMENTAL DESIGN:

See you at MSC 2015?

MSC 2015 is quickly approaching next month and we at RoosterBio are getting ready.  This conference is arguably the single most important conference related to MSCs, and Cleveland is the considered by many to be the birthplace of the current paradigm of MSCs used in therapeutic contexts.  We will be sending most of our company, and we do look forward to seeing everyone there. Not only is this conference full of great sessions and talks, but the networking at this bi-yearly MSC conference is always top notch and yet another reason to attend.

The faculty and sessions at MSC 2015 are hyper-relevant to today’s more important topics, and the sessions are organized by several key themes.  Day 1 of the conference will be kicked off with a Keynote from Arnold Caplan , the godfather of MSCs (and yes, if you Google “MSC Godfather” you get Arnold Caplan), who is always entertaining and insightful to where MSC technology is going. The sessions look to be focused on Clinical Trial updates by the likes of Athersys, Katerina LeBlanc, Dan Weiss and Jacques Galipeau, among others. 

Day 2 of the conference gets kicked off with a keynote from Frank Barry from The National University of Ireland at Galway, and he will be speaking on MSC Translation.  My favorite topic, MSC BioManufacturing, will be covered that morning, and we all know that MSC technology cannot be translated into humans without consistent, robust and cost effective manufacturing processes that are capable of maintaining the quality parameters and functions of these critical cells.  Sessions on MSCs in applications like cardiology and organ transplantation will follow, and the day will end with the session I am most excited about – Next Generation MSCs.  Jan Nolta and Mike West will highlight this “not to miss” session.

The final day of the conference will have a keynote from Stanton Gerson, followed by many new and impactful applications including MSCs in Cancer and Sepsis.  The last two sessions are on potentially the most impactful translational areas of MSCs (as it pertains with shear numbers of patients treated), which are the use in Sports Medicine and Veterinary Sciences.  I will bet that Bob Harman at Vet Stem has treated more patients with MSCs than any other clinic or company in the World – and I plan on asking him what that number is at the conference, so look for it in our Twitter feed.

It does look like the dedicated organizing team at Case has done a great job at organizing yet another stellar event, and we look forward to seeing you there.  Be sure to stop by our Booth and posters and say hello!