Center for Innovative Cancer Research, Ottawa Hospital Research Institute,
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Small molecules have historically dominated the pharmaceutical industry. Today, biologicals such as vaccines, therapeutic proteins, and monoclonal antibodies have quickly become a mainstay and are projected to constitute nearly 30% of total prescription sales for Big Pharma by 2017 . For vaccines in particular, growth is expected to be sustained by the development of emerging markets, novel vaccines for cancer and endemic diseases, as well as continued development and improvement of products and manufacturing capabilities .
Indeed, bio-therapeutics manufacturing is inherently complex and often a barrier to market entry. The necessity for using cells or even animaal tissues (e.g. embryonated eggs) to produce most bio-therapeutics puts significant constraint on both maximum capacity and process reliability. Virus-based therapies such as vaccines, oncolytic viruses and gene therapy vectors are complex macro molecules that can take in some cases nearly a month to amplify to sufficient quantities (e.g. Hepatitis A vaccine). For some therapeutics like oncolytic viruses and gene therapy vectors where therapeutic doses are often very high (>109 particles per dose ), manufacturing limitations can further compromise a product's commercial viability by limiting the maximum feasible therapeutic dose.
Process intensification technologies will undoubtedly play an increasingly critical role in overcoming these issues. Here, we define process intensification technologies as innovations that improve the efficiency of current manufacturing processes to make more product in less time, using fewer resources, which ultimately translates to a lower cost per unit produced. These process intensification innovations can be further subdivided into technologies that target upstream and downstream processes. Downstream innovations would include for example novel filtration or chromatography technologies that increase the recovery and/or purity of biological products. As these are ultimately dependent on the amount of product produced upstream, we will further focus on process intensification technologies that increase crude bio-therapeutic yields.
Cells Take the Limelight in Upstream Process Intensification
Many bio-therapeutics such as monoclonal antibodies and virus-based therapeutics are produced in cultured animal or insect cells. It is widely believed that higher cell density and healthier cells result in higher product yield. As such, most innovations in process intensification target methods to optimize or improve cell density and growth characteristics. This includes designer cell lines, media additives and novel bioreactors.
Designer cell lines
Use of continuously growing designer cell lines with improved properties over classically used substrates such as Vero, MRC-5, CHO, HEK293 and primary chicken embryonic cells has emerged as a strategy to improve both bio-therapeutic output and reliability. Well-known examples include PER.C6 (Crucell/J&J) and EB66 (Vivalis now Valneva). These suspension cell platforms grow to extremely high densities and are reported and advertised as being more productive for several applications including antibodies and viruses [4-6] One downside of this approach is the need to completely redesign the manufacturing process (e.g. switching from egg-based production to suspension cells), which makes using these designer cell lines less attractive for some well-entrenched bio-therapeutics (e.g. influenza vaccine).
The increasing regulatory requirement for animal-free production of human bio-therapeutics has led to a substantial rise in the use of serum-free chemically defined cell culture media. However, current chemically defined serum-free media often lead to lower production of bio-therapeutics. To counter this, cell culture media additives are widely commercially available to improve the growth of cells, which can significantly improve productivity. Common supplements include animal-free recombinant proteins such as albumin, transferrin, and insulin as well as plant-based protein hydrolysates. With the exception of a few formulations pre-tailored to specific cell lines (e.g. EX-Cell EBx for EB66 cells), custom optimization of media formulations for each cell line is typically required and is often an essential prelude to maximizing the production process of most bio-therapeutics .
Another important recent innovation of upstream process intensification is the latest generation of bioreactors. To this end, suspension cells have long been the standard in antibody production due to the availability and scalability of classical stir-tank bioreactors. Nonetheless, adherent cells can sometimes be more productive, or in other cases the only viable option. As a result various micro-carriers have been developed that permit the growth of adherent cells in stir-tank bioreactors. Newer bioreactor products, such as fixed-bed bioreactors have optimized this method for growing adherent cells at very high densities. For example, the iCellis Integrity® bioreactor (Pall Life Sciences) creates a remarkable 500 m2 of cell growth surface (~3000 roller bottles) on medical grade polyester microfibers packed within a 25L bioreactor that is not much bigger than a standard clothes washing machine .
Viral Sensitizer Technology: A New Upstream Process Intensification Solution Tailor-Made for Virus-Based Products
While most process intensification strategies have traditionally focused on the need to keep cells healthy and growing to high densities, to our knowledge no widely available technologies address other host cell mechanisms that can impact the production bio-therapeutics. In the case of therapeutic antibodies one can argue that innovations that improve cell growth and density also by default improve antibody yield. However, viruses are another matter. Given that viruses are intracellular parasites, viruses ultimately kill their host cells while cells generally work to destroy the virus. Indeed, the cellular antiviral immune response is a highly complex signaling system devoted to protecting cells, tissues, and organisms against viral infection. The viral sensitizer technology (VST) discovered and being developed in our laboratory targets this antiviral immune system and as a result enhances virus production.
VST encompasses a recently discovered and growing collection of small organic molecules that robustly enhance virus growth (in some cases >1000X) by broadly and effectively disrupting cellular antiviral defenses. While we initially discovered this technology in the context of oncolytic virus therapy , we have since found that VST is more generally applicable to a broad range of DNA and RNA viruses, substantially improving virus productivity. To this end, improvements in viral yields often in the order of 10+ fold can be obtained for viruses such as influenza and modified vaccinia Ankara (MVA) in typically used manufacturing cell lines (e.g. Vero, MRC-5, BHK21, chicken embryonic fibroblasts, MDCK).
Importantly, VST is compatible with cells cultured either with or without animal serum and leads to improved yields when using commercially available media supplements, with additive or synergistic effects in some instances. This is likely because classical media supplements aim to improve cell growth while VST compromises cellular barriers to virus infection, permitting more efficient viral growth and spread. In effect, this unique combination approach simultaneously caters to both the needs of the cells and the virus.
VST can also lead to impressive results when used in the context of fixed-bed bioreactors. Our experience with the iCellis Nano (smaller benchtop version of the iCellis Integrity) has been extremely positive to his end. To start, we have found that cell densities superior to what is typically observed in standard tissue culture can be achieved in certain contexts. Remarkably, using VST in this context led to virus yields equivalent to that obtained from several RC40 units (each 40 roller bottles) in a small toaster-oven sized bioreactor.
Process intensification is emerging as a key driver of the bio-therapeutics market. Technologies aiming to increase productivity are therefore highly desirable but have traditionally focused on maximizing cell growth and will eventually reach a plateau. Moving forward, new technologies such as VST that modulate key cellular processes to increase specific production efficiency of bio-therapeutics and that synergize with existing process intensification technologies will play an increasing role in the future.
About the Author
Dr. Diallo obtained his Bachelor's degree in Biochemistry at the University of Ottawa and a Master's in biochemistry at McGill University. He earned his Ph. D in Molecular Biology at the Université de Montréal and subsequently went on to do a postdoctoral fellowship at the Ottawa Hospital Research Institute (OHRI) where he worked on improving oncolytic virotherapy using small molecules. An Assistant Scientist at the OHRI, his research focuses on manipulating the cellular antiviral response using small molecules for a wide range of virus-based thera
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