Tony Butler
Manager of Single Use Systems
NewAge Industries-AdvantaPure
John Shyu, Ph.D.
Bioprocess Technical Services Manager
Corning Life Sciences
Dr. Nick Hutchinson
Market Development Manager
Parker Hannifin Manufacturing Ltd.
domnick hunter Process Filtration - Europe
Mark Stramaglia
Associate Director , Product Development, Life Sciences Solutions
Thermo Fisher Scientific
Donald Young
Senior Global Product Manager , BioProcess Containers, Life Sciences Solutions
Thermo Fisher Scientific
Cynthia Hoy, Ph.D.
Process Science Fellow, Life Sciences Solutions
Thermo Fisher Scientific
Dr. Jeri Ann Boose
Director, Biopharmaceutical Services
Eurofins Lancaster Laboratories
John Stover
Director of Product Regulatory Compliance
NewAge Industries-AdvantaPure
Todd Kapp
Market Development Manager
Parker Hannifin Manufacturing Ltd.
domnick hunter Process Filtration – Europe
1. What advances in the biotech industry have allowed for the implementation of fully disposable facilities?
Butler: The biotech industry, as a whole, has improved batch potency so that the average final batch size has decreased. Meanwhile the maximum working volume for singleuse bioreactors (SUBs) and single-use mixers (SUMs) has increased to 2,000 liters. This confluence of larger equipment and, more importantly, smaller batch sizes, has enabled disposable facilities.
Shyu: In recent years, surges in the availability of disposables and single-use equipment and technology have changed the landscape of the biotech and pharmaceutical industries. With such a wide range of disposables and single-use equipment now available, each facility is able to select products that are optimized for their unique production environment. Manufacturing companies like Corning Life Sciences, constantly seek out new technologies and products that can reduce the complexity of their production processes. The introduction of disposable containers and novel high yield cell culture vessels that can fully operate at the level of large fermenters and steel bioreactors has helped to drive cost efficiencies, to some extent reducing capital expenditure on equipment and production space.
Additionally, many of the new plastic-based disposable products are flexible in nature, and are able to be used for many different applications in both the biotech and pharmaceutical industry. This can also lead to further savings in both investment and operational costs. Lastly, not only are novel technologies more compact in nature, but the biological tools and technologies, such as optimized cell culture surfaces, are more effi cient as well. High producing cells are compensating for earlier low productivity cells, allowing facilities to incorporate more disposable products in the production line.
Hutchinson: Changes in both biomanufacturing processes and advances in single-use technologies have allowed for the implementation of fully disposable biopharmaceutical manufacturing processes from thawing a vial of cells through to filling a vial of purified product. Cell lines capable of expressing product titres of 10 g.L-1 produce the same total mass of product in a single-use 1000L bioreactor as cell lines from a decade ago that produced in 10,000L stainless steel bioreactors at titres of 1 g.L-1. The specificity of these medicines which is their great benefit in minimizing side-effects while maximizing efficacy does, however, constrain the size of the patient population which benefits from treatment by any given product thereby limiting batch-sizes. Smaller batches are considerably more amenable to processing with single-use technology than those requiring kilo quantities of protein for filling.
Technological developments have made single-use technologies mainstream, viable processing options. Suppliers are implementing ever more sophisticated methods for ensuring quality and security within supply chains providing end-user quality departments with the confidence they require for full implementation. The developments of robust sensing and automation solutions within single-use equipment is providing biomanufacturers with the control they need to consistently manufacture safe and efficacious product.
Stramaglia: Ten years ago, everyone was talking about not having enough bioreactor capacity to make biotherapeutics. Since then, several improvements in fed-batch culture have set the stage for smaller scale disposable processes to be successful:
- Improved cell line engineering – it is now typical to get over 1g/L titers for antibody production in systems like the Freedom® CHO-S® Kit
- Base medium improvements allowing for greater than 10M cells/mL peak cell densities (e.g., Gibco® CD FortiCHO™ medium)
- Integrated feeding supplements extending cell viabilities and increasing production outputs by multiple-fold (e.g., Gibco® CD EfficientFeed™ C supplement)
- When that is not enough to maintain specific productivities for maximum output, functional additives have become available to maintain specific productivity levels (e.g., FunctionMAX™ TiterEnhancer)
With those improvements, it is common for a final process to get 3-5g/ L production which allows use of smaller bioreactors more in the range of disposable bioreactors at 1000-2000L. What is still needed are ways to assist making the process set up even simpler and more efficient such that these smaller runs in disposable systems can become the routine standard for all forms of biotherapeutic production.
Young: Recently, we have seen a growing demand for single-use biopharmaceutical facilities, due to the increased requirement for flexibility, process efficiency and speed to market. There is a desire to minimize timelines and financial risks. “Biotech-on-demand”, the ability to quickly support local manufacturing capacity, meets market needs and potentially National Security needs to easily and rapidly respond and protect the public from large-scale, fast moving epidemics and pandemics.
From a manufacturing process point of view we have now seen the development of high yielding mammalian cell line expression systems that routinely produce therapeutic proteins in the grams/ liter range. This increase in titer has reduced the industry’s reliance on large-volume bioreactors above 2000L. The outcome of this is the option to scale-out instead of scaling-up. Manufacturers can now produce commercial-scale product volumes utilizing several 1000L or 2000L bioreactors instead of a single process train up to tens of thousands of liters.
Single-use systems drive many cost savings as they do not require clean-in-place and steam-in-place operations and systems, which are expensive to install and maintain, consume tremendous amounts of water and generate significant amounts of liquid waste. These systems also help satisfy sustainability or “green” initiatives. The product breadth of upstream and downstream single-use products continues to expand to include single-use containers for production and storage of process liquids for sampling systems and for harvesting, dispensing, storage and transport of bulk drug substances.
2. Discuss emerging process development technologies that contribute to improved cell lines.
Butler: On-line real-time monitoring and control of glucose levels in cell batch process cell lines is an emerging technology. Knowing the optimal glucose level and maintaining this level for cell lines will improve cell growth and viability.
Shyu: The way cells are grown has changed substantially in recent years. As improvements are made to cell culture techniques, the introduction of smart vessel technologies, combined with optimized surface treatments, has also contributed to improvements in cell line development and in production. The ability to monitor growth conditions and to detect byproducts has allowed for better cell selection and expansion dynamics.
One of the most addressed process development technology trends is the optimization of cell culture process scale-up that focuses on high-density production of quality cells. As we further increase our knowledge of cellular physiology, we incorporate surface chemistry and engineering technologies to create the most beneficial biological growth systems suitable to production of viable cell lines.
Corning recently developed the unique HYPER technology culture vessels. This novel technology introduced the concept of breathable gas-permeable membranes in multi-layer cell culture vessels which reduce the footprint required to achieve a specific production level. Corning also incorporated CellBIND®, a modified and improved tissue culture treatment, into the HYPER culture vessels. Novel tissue culture treatments such as Corning® CellBIND® and Corning® Synthemax® (a synthetic alternative to biological coatings) can enhance cell attachment to improve cell growth conditions and recovery viabilities.
Hoy: Since the commercial launch of tissue plasminogen activator in 1986, Chinese hamster ovary cells (CHO) remain the workhorse for the bio-production of mammalian derived human therapeutics as these cells have produced the majority of nearly 100 approved products. Availability of clear commercial licensing strategies, well characterized cell banks, scalable processes, and robust, rapid growth in bioreactors using animal-origin free raw materials contribute to the popularity of various CHO strains as the host cell of choice.
Molecular characterization assays with high resolution and precision have identified required protein quality attributes, and together with genome sequencing data now available for certain lines (DG44, CHO-K1, and CHO-S® Cells), have elucidated the requirements for post-translation modifications and have paved the way for genetic modification and engineering for host cells for relevant attributes such as, for example, glycosylation potential. Identification of metabolic pathways from genome sequencing has allowed further optimization of nutritional support for high cell densities, productivity, and modulations of protein attributes to achieve the correct biotherapeutic profile. Technologies to deliver key nutrients in increasing concentration to support these high density cultures have been integral factors in the success of these advances.
Improving expression vectors has long been a focus, but achieving good expression with accessible licensing terms, as with the Freedom® CHO-S® Kit is a recent advancement and may substantially help reduce the overall costs of a development program. Tools such as the Clonepix FL facilitate automated, high throughput screening and expansion of thousands of clones. Characterization of large numbers of clones in a fed batch bioreactor-like setting using automated micro-bioreactors promises to identify scalable clones faster. Still posing a challenge is clone stability, but recent advances in site specific integration are promising.
3. Discuss the components of effective detection for adventitious agents.
Shyu: One of the most critical challenges facing any manufacturing process is the assurance of products that are free from adventitious agents. Stringent and systematic approaches should therefore be taken throughout the production process to effectively screen for the presence of such agents. Although various methods exist to detect adventitious agents, each individual method has limitations in terms of specificity and sensitivity. Thus, using a combination of approaches and methods of in vivo and in vitro tests, along with high sensitivity biochemical assays and molecular techniques, far outweighs the assurance of any single approach or method.
It is also important to keep in mind that the methods and approaches used to detect adventitious agents must be constantly validated as these can evolve over time. Each day, novel viruses and substrate agents are being discovered and will likely continue to be discovered. Improvements in detection technology are constantly on the horizon and can be a powerful means to support adventitious agent safe product development and manufacturing.
Boose: Biopharmaceuticals from animal and plant systems have a risk of contamination from bacteria, mycoplasma and viruses. Such contamination may come from the source materials and/or the raw materials used during manufacture, or it may come from the adventitious introduction of these agents during the manufacturing process. Testing of the source materials and raw materials for adventitious agents is the first tier of a three-tiered approach toward preventing contamination of biotechnology products. The second tier assesses the capacity of the production process to clear adventitious contaminants of concern, and although this is most frequently done for viruses (viral clearance studies), it may be done for any contaminant of concern. The third tier involves testing of the product at the appropriate stages of production for the absence of adventitious contaminants.
With regard to bacteria and mycoplasma, the testing performed on the source materials, raw materials and product are the compendial sterility/bioburden and mycoplasma assays. There are numerous assays available to detect viral contaminants, including both broad-based viral screening assays (in vivo and in vitro), as well as assays for specific viruses that are typically based upon the use of immunological and molecular assay platforms.
4. How do you tackle challenges associated with raw materials?
Stover: The disposable elements of a single-use system are made mostly with various polymers. Once the components needed are identified, a thorough study of the chemical characterization of each polymer should be performed. These include extractable and leachable analysis, chemical compatibility with all intended process and product fluids, and environment and process impact that includes worst case time and temperature exposure. Ideally, these assessments should be performed on actual finished components to ensure that the manufacturing processes of the components are accounted for. It would also be recommended that these studies are performed on components after they’ve been exposed to the intended sterilization technique.
Shyu: The availability of raw materials for any manufacturing company is always a topic of discussion and debate. How to secure raw materials can be a challenge, especially in an everchanging market. Strong global economic growth driven by rapid emerging markets can greatly affect the availability of raw materials and set unexpected price swings.
To truly be on top of the challenges that can be associated with raw materials, the best strategy one can set in place is an adequate monitoring system of how and where your raw materials are coming from. The ability to secure supplies of raw materials is critical to manufacturing success. Having the right framework conditions to monitor your primary source of raw materials, together with securing an alternative source, will allow you to predict and potentially be ahead of any unexpected challenge in availability
Kapp: Parker domnick hunter actively participates in the leading industry organizations, principally the BPSA, which drive standards in single-use technology. These organizations are helping to create guidelines for raw material suppliers to follow and utilize. This is important because supply chains of consumable components used in bioprocessing are getting longer and the ability of the biomanufacturer to control these more challenging. Parker domnick hunter works collaboratively with our suppliers to ensure we keep pace with single-use bioprocessing requirements and update applicable quality controls such as incorporating the principles of Quality-by-Design. As part of this collaborative approach, we mutually agree upon specifications with raw material suppliers including agreement related to change notifications. Our preferred suppliers are those that meet our requirements totally or are working toward that goal. In addition we are working with our suppliers to create relevant data packages for a customer’s unique application. Finally as a responsible supplier to the Biopharm industry we audit our raw material suppliers’ facilities and their quality management systems.
Boose: Raw materials have been linked to many mycoplasma and viral contamination events in the biopharmaceutical industry, making it very important to assess raw materials for their risk for introducing these contaminants into the process. A key component of a good raw materials program is supplier management. It is recommended that a vendor quality audit be performed with an emphasis on pest control and cleaning validation, as well as a focus on the manufacturing process for each individual raw material. Each raw material should then be characterized as low, medium or high risk for the possibility of introducing contaminants into the process. A risk mitigation strategy for individual or strategic combinations of raw materials should then be developed. This strategy can include recommendations with regard to safety testing of the material prior to use, the addition of adventitious agent inactivation/removal steps to the manufacturing process for each raw material and/or the addition of inactivation/ removal steps immediately prior to use of the raw material in the manufacturing process. Commonly used inactivation procedures include, but are not limited to: exposure of the raw material to high temperatures for short periods of time (HTST), gamma-irradiation, and monochromatic UVC treatment. Although zero-risk from raw materials is not possible, the multi-faceted approach just described reduces the risk to the lowest possible level.
Young: For single-use products, we address raw materials supply through a business-wide commitment to quality. This begins with a detailed scrutiny of component suppliers, continues with the design and materials used in construction of those components, includes their associated manufacturing processes and extends to the delivery of our finished product.
It is critical to manage the supply chain and traceability of the raw materials through a material and component testing program and strict adherence to our approved supply program. This commitment to quality includes highly qualified and well-trained staff, a strong technical/materials science team and ongoing engineering team support. In combination, these measures steer our selection of suppliers, selection of component design, and our selection of materials of construction to achieve the highest level of quality.
5. Walk us through how viral clearance barriers mitigate risk in manufacturing processes.
Shyu: Today’s manufacturing processes of biological products involves high levels of structural complexity and stages that can pose unique challenges in providing products that are of high quality and with the utmost safety. Determining the best critical process parameters and implementation of critical control points can nevertheless improve both the quality and safety of biologicals. It is therefore important to establish concise detection system profiles along with virus inactivation and removal methods in order to mitigate any risk during the manufacturing process.
Proper incorporation of virus barrier techniques both upstream and downstream can minimize the risk of any manufacturing processes. Mitigation processes should start with treatment of raw materials using technologies such as γ-irradiation or UV-C inactivation to ensure a pro-active approach against virus contamination. Further down in the production line, virus filtration units are often included for additional levels of viral safety. Lastly, proper and relevant screening assays should be incorporated during the final production steps to ensure overall safety of deliverable products. Any barriers implemented must be critically explored and the robustness of the operation demonstrated to limit any potential spread of contamination
Boose: The ability to detect a low-level viral contaminant in source materials, raw materials and product intermediates is limited by the direct testing methods used for this purpose. Specifically, direct testing can be performed only for known viruses for which assays systems are available, and the assays are limited by their sensitivities. Viral clearance studies are a complementary strategy to the direct testing of materials and product and are considered to be the “what if experiment”. The viral clearance study complements the direct testing approach by asking the following question: Will the manufacturing purification process be able to inactivate virus or separate it from the product in the event that a low-level viral contaminant is present, but not detected during the testing of materials and product intermediates? Viral clearance studies have two goals that are attained through the use of a strategic panel of spiking viruses. Viruses on the panel are selected to (1) represent those viruses that might be expected to contaminate the starting materials and (2) challenge the process with viruses having a wide range of physical and chemical properties. If appropriate levels of clearance are demonstrated for the full virus panel, the clearance study accomplishes the dual goals of demonstrating that the process has the ability to clear both expected viral contaminants as well as the novel or unpredicted viral contaminant.