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 . 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.)
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 . The FBS should be processed under current good manufacturing practice (cGMP) standards that set minimum requirements for the facilities, materials and protocols used . 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 variability.
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 , 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) . 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  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 . 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 .
Although strict standards exist for the source and quality of FBS, the exact chemical composition (i.e. proteins, growth factors and hormones) of each batch has remained unknown/undefined. Batch-to-batch composition variability has the potential to influence cellular behavior, phenotype and growth performance [2,14,15]. Serum qualification studies typically test multiple batches of FBS over multiple donor cells to determine the optimal serum lot for cell manufacturing . Some serum providers characterize levels of major components in specific FBS lots and offer “lot matching” and “lot reservation” services, but this is limited as serum volume (i.e. lot size) requirements increase. While small-scale clinical studies can be conducted with a single batch of FBS, both supply chain and performance challenges increase when attempting to scale up production for large-scale clinical trials and commercial cell manufacturing. The ability to reserve large lots of batch-tested FBS is needed as hMSC-based therapies become commercialized.
Serum is a byproduct of the beef and dairy industry. As a result, the supply and cost of serum is dependent on external factors such as outbreaks of disease in cattle herds, the price of beef and dairy and the price of cattle feed. Current supplies of GMP-grade FBS will not be able to support the expected rise in demand due to the initiation of large-scale clinical trials and introduction of commercial cellular therapeutics. We are reaching a “peak serum” state where demand for serum from the cellular therapeutics industry is exceeding the maximum achievable production level . This has led to an increase in cost of GMP-grade serum, with a 3-fold price increase between 2009 and 2012 . Furthermore, FBS is one of the most expensive forms of serum, as multiple calf fetuses are required to create one liter of serum . The Stem Cell Assays blog described FBS as one of the most expensive raw materials and a major driver in the cost of goods sold for cellular therapeutics. Thus, it has been suggested that transitioning to FBS alternatives will likely be a function of supply and cost issues more so than regulatory concerns .
Addressing the peak serum challenge
Several alternatives to FBS have been explored including human platelet lysate (HPL) and chemically defined, serum-free medium (SFM); these are two of the most common clinical alternatives to FBS .
HPL: HPL has been shown to induce a higher proliferation rate of hMSCs compared to FBS, due to a rich concentration of a variety of growth factors [16,17]. However, similar to FBS, its chemical composition is poorly defined and pooling of donors is used as a strategy to address lot-to-lot variability. Additionally, allogeneic HPL holds the risk of carrying human pathogens requiring extensive (and expensive) safety testing prior to use. Autologous HPL has expected performance variability and supply limitations due to donor issues . To date, there has not been a thorough supply chain analysis performed to estimate the amount of HPL that would be available for Cell Therapy products brought to market using HPL as a raw material.
SFM: SFM is favorable due to its defined and consistent chemical composition and reduced risk for disease transmission. However, SFM formulations are often not able to elicit consistent biological function of hMSCs or support consistent cell proliferation in different cell culture environments, including during scale up manufacturing [6,18]. This suggests that SFM formulations need to be optimized for every cell source and culture condition involved in a specific protocol, a process requiring an extensive amount of time and money . While SFM will be the long term solution for clinical-grade hMSC production, there are still several technology and business challenges to address prior to its wide-spread implementation.
Hybrid strategies: One middle-of-the-road strategy is to combine serum and serum-free culture steps in the cell manufacturing process. Using serum during hMSC isolation and Master Cell Bank production, but subsequently transitioning to SFM for final therapeutic production is a documented and commonly-used technique that mitigates the aforementioned challenges to serum use . This strategy could significantly reduce the need for FBS (up to 99%) in clinical and commercial Cell Therapy manufacturing processes . Alternatively, RoosterBio has taken the approach of minimizing serum use by engineering a rich culture medium, similar to a chemically-defined SFM medium, supplemented with very low levels of high-quality serum to stabilize the formulation. Coupled with a streamlined manufacturing process, this approach reduces serum requirements by well over 90%, greatly extending the lifetime of qualified serum lots and bringing consistency to RoosterBio’s cell culture media products. Thus, RoosterBio hMSCs display consistent growth rates and functional characteristics across cell and media lots and our cell-media systems are amenable to scale-up manufacturing processes.
The Future of MSC Clinical Translation
The current state of hMSC clinical translation requires researchers to invest in their own, individual media development and cell culture protocols; an expensive process that requires very specific skill sets and takes years to develop. What the MSC World needs is a simple solution that can generate cells quickly and consistently, or an off-the-shelf hMSC product that can be used on-demand, no cell culture required. In the 1980s, growth factors underwent a similar transition to becoming simple, thaw-and-use reagents, with advances in recombinant DNA technology [19, 20]. When a similar solution becomes commonplace in the coming years, we will see a new paradigm in the manufacture and widespread use of hMSC-based cellular therapeutics.
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This blog was first published in Democratizing Cell Technologies, A RoosterBio Blog, written by Michelle S DiNicolas, PhD, – http://roosterbio.blogspot.com/2015/10/the-use-of-animal-serum-in-clinical.html