Single-Use Operational Excellence Explained: Effective Lifecycle Management

Single-use technologies (SUTs) have introduced a broad range of cost and operating efficiencies to bioprocessing operations. Both in upstream and downstream, the technology offers new flexibility — but managing operations effectively can be challenging without a deeper understanding of the single-use life cycle and the effective role SUTs can play throughout biomanufacturing operations. 

For most of the biopharmaceutical industry, the processing of large molecule therapeutics of all kinds has traditionally been tied to proprietary large-scale stainless steel systems featuring miles of stainless steel piping, fixed holding tanks, mixers, bioreactors and cleaning equipment. Although fixed systems offer their own operational economies, especially at commercial scale, biopharmaceutical manufacturers are seeking flexible process solutions to help them better respond to the changing business, product, financial and regulatory circumstances facing the industry today.

However, with the advent of single-use technologies (SUTs), biopharmaceutical manufacturers now have a viable, affordable path to introduce flexibility and new operational economies into bioprocessing operations.

Single-use technology is not applicable to all molecules or bioprocess steps and, in and of itself, will not assure a better product, improve margins or make a more competitive product. However, SUTs can deliver a range of benefits if assessed thoroughly before implementation. For better outcomes, a lifecycle approach to the assessment can help an organization transition successfully to SUTs and realize the benefits of the technology. Best practice puts the assessment process in front of a cross-functional operations team to review and understand the complete manufacturing process as it relates to adopting and integrating SUTs.

A Singular Mindset for SUT Assessment

Instead of looking at individual components and assemblies, real value comes from looking at a given process with a broader perspective, examining operations comprehensively for interactions and adjacencies, not only with components and systems, but throughout operations and with cross-functional stakeholders. Following are key elements that can help frame an effective assessment program and define a sustainable single-use lifecycle for a given process and plant setting.

For better outcomes, a lifecycle approach to the assessment can help an organization transition successfully to SUTs and Realize the benefits of the technology.

Validation Planning (Qualification, Commissioning and Validation)

To validate a given manufacturing process and be compliant, a biopharmaceutical manufacturer must submit to regulators an overall master plan. This plan covers plant, equipment, process, personnel and documentation, including design (DQ), installation (IQ), operation (OQ) and process qualification (PQ) elements that support the plan. Design qualification associated with conventional, stainless steel systems has typically taken place prior to the construction of the equipment. Single-use technologies, however, offer the ability to decouple some DQ activities, like material compatibility, because SUT materials may be prequalified. Single-use equipment is often less complex than conventional stainless steel counterparts. This simplicity offers an opportunity to reduce the effort and time associated with IQ and OQ.

Training for Excellence in SUT Operations

Compared with conventional fixed-pipe stainless steel systems, SUTs will require fresh training and an alignment of operations to suit the more intensive reliance on operators for set-up, installation and use. Bear in mind operators are not the only functional group to be addressed. Single-use technologies introduce a whole new supply and inventory management aspect to operations, and warehouse/material handling personnel will be impacted.

Effective training is critical to sustaining the operational efficiencies associated with SUTs. New routines and training should be introduced to address both the mechanical and material intricacies of SUT systems and the operational procedures to keep operations functioning at optimal levels.

Assessing Operations

The transition from process development to manufacturing scale involves managing intensive change, typically in operating spaces (classification), layout (interconnected, adjacent unit operations) and personnel (type and training level). The efficient flow of material, personnel and waste through the manufacturing environment is critical to effective operations and its ability to preserve the integrity of the manufacturing space. Single-use components and assemblies are involved in most, if not all, process steps and the volume of SUT materials introduced into the manufacturing space is significant. As such, staging, use and disposal of these items are central to properly aligning manufacturing’s material and waste workflows.

Institutionalized standard operating procedures (SOPs) are necessary to formalize the activities and ensure a robust manufacturing process. Within the manufacturing space, SOPs should include contingencies for single-use component/assembly replacement, or substitution, and reinforced with training. Materials inventory, transfer and record-keeping should not be overlooked either. 

Design and Documentation

In the biomanufacturing suite, the process train forms an integrated manufacturing line with all the necessary unit operation and support equipment. Unlike manufacturing, process development is focused more on technical performance, rather than equipment integration and overall, integrated manufacturing process operations.

Process development activities can offer an appropriate proving ground for specifying single-use assemblies to suit specific unit operations. Integration, or more specifically, the interconnection of various adjacent unit operations that make up the manufacturing process using single-use assemblies, requires a thorough understanding of the available space and layout as well as specifics associated with connections and logistics, product transfers, etc. At this point documentation requirements can also be determined for the drugs manufacturing program.

Single-use technologies introduce a whole new supply and inventory management aspect to operations, and warehouse/material handling personnel will be impacted.

Sourcing and Procurement

Unless a drug maker intends to design and manufacture single-use elements in house, supply chain partners are required. Finding qualifiable SUT suppliers is paramount and critical to secure a reliable supply. Representing the internal stakeholders, the sourcing function must be able to communicate the appropriate business and technical requirements, externally, to potential suppliers. Single-use system design, unit quantities, delivery timelines and documentation requirements are a few of the common considerations. Single-use technology has also increased the interconnectivity of the supply chain. Supply chain transparency is important because buyers often source components, and semi-finished and finished assemblies, from the same lower-tier suppliers used by other top-tier suppliers within the supply chain. With a focus on the drug manufacturing process, it should be clear as to the state of an assembly’s design: prototype versus final released version.

If any design and review steps remain, procurement plans must reflect this uncertainty. Using a supply agreement to summarize/catalog and codify the quality, commercial, technical and documentation aspects of the single-use lifecycle will go a long way toward keeping individual yet interdependent businesses aligned.

Continuous Improvement Ahead

Real-time data, operator feedback and input from the supply chain contribute to a more functional, efficient single-use lifecycle. In every aspect it is important to consider the internal and external stakeholders involved in the SUT continuum, and work to promote communication among all parties to support sound, GMP-compliant operations and continuous improvement over the long term. 

Conclusion

Single-use technologies, inclusive of components and assemblies, have become an effective means for many biopharmaceutical manufacturers to achieve improved product quality, greater plant utilization, and overall operational effectiveness. When implementing SUTs, the biopharmaceutical industry has come to understand that the greatest benefits come to those who have analyzed their end-to-end biomanufacturing operations comprehensively and have defined a single-use lifecycle best suited to their products, process and organization.  

Originally published on PharmasAlmanac.com on October 26, 2017.

Stainless Steel to Single-Use: How to Adapt to the Changing Landscape in SUT

Avid Bioservices began transitioning to single-use technology (SUT) several years ago and has reaped considerable benefits, both to our operations and to the agile service we provide customers.

Saying Goodbye to Stainless Steel 

Stainless steel facilities are on their way out. With the high titers obtained today, 2000-L single-use (SU) bioreactors are sufficient for most processes, and some vendors are developing systems up to 5000 L. When they are available, the range of SU bioreactor sizes can support the vast majority of biologic products on the market and in development, except perhaps global blockbusters.

Avid at the Forefront

Avid Bioservices was one of the first adopters of large-scale single-use technology. The decision to move to 1,000-L disposable bioreactors was made in 2007. However, at that time, the company had years of experience with SU biocontainers for buffer and intermediate product storage and single-use unit operations such as membrane chromatography. 

Offsite Fabrication for a Reduced Construction Timeline

Avid’s Myford facility, designed to exclusively use SUT, was completed in 2016. The facility is truly a building within a building, with modular walls, ceilings, and many other components fabricated offsite and then installed within an existing building. There was no need for extensive external renovation, and this approach significantly expedited the construction process from groundbreaking to validation and facility start-up. 

Access to Upstream Scale-Down Models

Unlike with stainless-steel reactors, scaled-down models of larger SU bioreactors are available from most vendors. These smaller systems (3L, 50L, 200L) are designed to mimic performance at larger scales, allowing process development to readily advance from the lab to the pilot plant and, ultimately, to production. While some gaps and issues remain, access to these scaled-down models confer a significant advantage on disposable technology.

Simplifying Investigations

Product quality investigations involving stainless-steel reactors are often incredibly complicated when all of the equipment and systems involved need to be examined or tested. SU bioreactors are inherently less complex and allow manufacturers to work with the SUT vendors to test the materials involved and help determine root cause. This simplification can enable faster investigation turnaround times, reduce investigatve costs, and prevent extended equipment tag-outs. 

Constrained by Availability

As with any production system, SUTs have their own limitations. One of the biggest constraints is the low availability of different technologies. Only a few vendors offer bioreactors, tubing assemblies and other consumables have been limited until recently, and prepacked SU chromatography columns remain smaller than what would be ideal for commercial manufacturing. Additionally, only a few SU sensors are available, and considerable work remains to optimize their performance. 

Users of disposable technology must also consider the consumables associated with SU equipment as critical raw materials. They are reliant on consumable vendors to ensure that production remains on schedule, which carries measurable risk. Vendors are impacted by natural disasters, including pandemics like COVID-19. Disrupted supplies, absent dual sourcing, or inadequate crisis plans can have a tremendous impact on the ability to continue manufacturing critical drug substances and drug products. 

There are also concerns regarding the availability of testing data with respect to the integrity of biocontainer films, compatibility with various biologic and chemical compounds, and extractables and leachables (E&L). The largest vendors have been proactive in providing data packages to assess these issues, but all possible application scenarios cannot be evaluated in advance. 

A Wish List for Vendors

Going forward, it will be critical for vendors to develop products that address the limitations of existing systems to boost flow rates and increase productivity, such as larger-diameter prepacked chromatography columns and larger and more efficient membrane filtration systems for harvesting and final product filtration. Standardization of the user interface for different systems would also greatly simplify things for operators. 

Transforming Avid’s Offering

As a multiproduct manufacturer, there are clear advantages to SUT over stainless-steel technology, going well beyond eliminating the need for cleaning and cleaning validation, and reducing risks of cross-contamination. SUTs offer more flexibility and accelerated time to market with straightforward scale-up of upstream processes, simplified investigations, and much shorter setup and changeover times. With disposable systems, Avid produces products more quickly, expediting our ability to bring in processes, scale them up, and deliver products to customers and ultimately to patients.

Originally published on PharmasAlmanac.com on December 9, 2020.

Evaluating Single-Use Technologies for Biomanufacturing

Single-use technologies (SUTs) are ideal for multiproduct facilities producing smaller volumes of materials. In addition to minimizing the risk of cross contamination, they save time and money with respect to setup and switching from one product to another. If current supply constraints are not addressed, however, the advantage provided by SUTs may be dramatically reduced.

Growing Adoption of Single-Use Technologies

Interest in biologic drugs was already increasing before the COVID-19 pandemic, and growth of this market has only accelerated as companies have sought to develop vaccines and therapeutics targeting the SARS-CoV-2 virus. The need to rapidly deploy manufacturing capabilities for these products has dramatically increased the implementation of single-use technologies (SUTs) on the commercial scale. Outside of COVID-19 treatments, many of the new biologics are targeted therapies aimed at treating smaller patient populations. As such, smaller volumes of drug product are required, and SUT solutions are often preferred for new facilities constructed today. 

As a result, Markets and Markets predicts that the value of the global single-use bioprocessing market will increase at a high compound annual growth rate (CAGR) of 20.5% from $8.2 billion in 2021 to $20.8 billion by 2026.1 Demand for single-use (SU) bioreactors, meanwhile, is expanding at a CAGR of 13.5% and will surpass $1.5 billion by 2026, according to Transparency Market Research.2

Choosing Single-Use vs. Stainless

There are certain conditions ideally suited to SUT implementation. SUT is most widely used for biologics production but rarely implemented for the manufacture of chemical APIs due to the potential for more interactions and degradation of the plastic biocontainers and other components.

Until recently, 2000 liters was the maximum single use bioreactor (SUB) available on the market. Today, that has increased, with SU bioreactors up to 6000 L now available, with the first 6,000-L SUB installed at a new contract development and manufacturing facility in China in 2020.3 Few SUBs of this scale are in use, however, with most manufacturers electing to stick with SU bioreactors of 2000 liters and smaller.4

Because SUT eliminates the need for cleaning and cleaning validation, as well as steam sterilization and all of the supporting infrastructure, this greatly minimizes the risk of cross-contamination. This makes SUT ideal for multipurpose facilities producing many different lower-volume products and frequently switching between products in a given bioreactor. For established high-volume products that require numerous batches per year, stainless-steel (SS) bioreactors are often still preferred.

As long as SU supply is not constrained, the time and cost for establishing new SU facilities are much less than those for plants dedicated to stainless-steel bioreactors. However, the availability of SU components is currently a concern given a confluence of current events including: supply disruptions caused by shutdowns and reduced production as the result of the COVID-19 pandemic; the dramatic increase in SU implementation by COVID-19 vaccine manufacturers at commercial scale; and COVID-19 projects receiving top priority above all others, meaning SUTs are in high demand.

SU Scaling

The elimination of cleaning processes between products simplifies the scaling of SU bioprocesses. The need to demonstrate equivalent cleaning through the cleaning validation process as processes are scaled to larger bioreactors is eliminated. As a result, scaling can be accelerated in SU systems compared with SS, which results in a faster process development timeline. It should be stressed, however, that other scaling issues related to the impacts of bioreactor size on key process parameters — power inputs, mixing gas transfer, feed rates, etc. — must be addressed when scaling in both SU and SS bioreactors.

More than Bioreactors

In addition to SU bioreactors, SU biocontainers are also available for media and buffer preparation and for storage and transfer of bioprocess fluids. In addition, integrated SU assemblies are now available for most downstream processes, from chromatography to tangential-flow filtration. In addition to traditional filter solutions, which have always been SU, suppliers have developed SU membrane technologies that provide elegant solutions with improved performance over older, traditional filters.

The ability to employ SUT across the entire upstream and downstream workflow dramatically simplifies process setup, due to the elimination of any cleaning and cleaning validation requirements. Similarly, switching from one product to another in a multiproduct facility is facilitated for the entire process.

Going SU for Clinical Manufacturing

Scorpion Biological Services is an integrated contract research, development, and manufacturing organization (CRDMO) focused on cell- and gene-based therapies and large-molecule biologics. We provide a broad array of biologic manufacturing, analytical, and R&D services from our San Antonio, Texas, facilities using American equipment, reagents, and materials.

Our new clinical manufacturing facility in San Antonio will be fully SU, other than utilities and a SS autoclave system, with SU bioreactors with a capacity of up to 2000 L.  

Managing Supply Uncertainties

The Scorpion clinical manufacturing facility is planned to be operational in mid-2022. Scheduling and planning can be challenging given the current supply constraints. We have ordered millions of dollars of SU stirred-tank supporting equipment that will be installed months before our SU bioreactor bags arrive, as the latter will not be available for nearly one year. The goal is to have preordered everything so that materials arrive and are in inventory when we are ready to be up and running.

References

  1. Single use Bioprocessing Market by Product (Media Bags and containers, Bioreactors, Mixers, Assemblies), Application (Cell Culture, Mixing, Storage, Filtration, Purification), End User (Biopharma Companies, CROs, CMOs) – Global Forecast 2026. Markets and Markets. Aug. 2020.
  2. Single-Use Bioreactors Market.Transparency Market Research. 1 Dec. 2017.
  3. ABEC To Deliver Industry’s First 6,000 Liter Single-Use Bioreactor to BioInno Bioscience. 28 Oct.  2020.
  4. Niemic, Charlotte. “Throw-away Culture,” com, 17 Apr. 2019.

Originally published on PharmasAlmanac.com on November 22, 2021

Accelerating the Development of Viral Vector Manufacturing Processes

INDUSTRY LEADER INSIGHT: Viral Vector Manufacturing

Not Your Traditional mAb Process

While lower molecular weight biologic drug substances are often produced via fermentation, larger recombinant proteins and monoclonal antibodies (mAbs), which account for the largest fraction of biologics on the market today, are generally manufactured using well-established platform processes. As a result, production equipment has been designed for mAb manufacturing, and this space is well serviced by equipment suppliers.

Viral vectors are substantially more complex than recombinant proteins and mAbs, and very different biology is involved in their production. For instance, viruses often kill the cells that are used to produce them, which creates complications when scaling processes. Viruses are also substantially larger than recombinant proteins and mAbs, and are also highly charged. 

Consequently, the equipment and reagents used for mAb manufacturing may not be optimal for the production of viral vectors. While some aspects of the technology have applicability, very different culture formats  most notably adherent cell culture in plasticware  have typically been employed for viral vector development and clinical trial material production.

Brammer Bio has leveraged hardware technologies developed by Pall to provide enhanced services to its clients that require the scale-up of viral vector manufacturing processes.

Suspension vs. Adherent Cell Culture

In suspension cell culture, the cells are free floating in the culture medium, while, in adherent cell culture, the cells are attached to a substrate in a monolayer. Adherent cell culture is used for certain cells, including cell lines used for viral vector production that must be anchored in some way to enable cell survival. 

Traditionally, suspension cell culture has been performed in stirred-tank bioreactors, while adherent cell culture has been achieved using roller bottles, flasks and plastic flatware, such as Corning’s HYPERStack® or Nunc™ Cell Factory™ vessels. Indeed, the majority of viral vectors in the clinical pipeline were initially produced via adherent cell culture, and an extensive knowledge base has been developed around the optimized production of viruses in this manner.

Suspension cell culture formats have been developed for the manufacture of adenovirus (AV), adeno-associated viral (AAV), retroviral (RV), and lentiviral (LV) vectors in HEK 293 cells and other cell types. Suspension culture using insect cell systems has also been applied to the production of AAV vectors (Figure 1).1 While these processes are scalable, the level of process understanding can be limited.

From Plasticware to Bioreactors

The challenge with adherent cell culture is the lack of scalability afforded by these processes. Production of large quantities of viral vectors on plasticware requires scale-out (vs. the ability to scale-up). Cost scales directly with the addition of more flasks or trays and more plasticware also takes up a larger footprint in the plant. These processes are highly labor-intensive, and scaling-out introduces the need for multiple rounds of manipulations, which can lead to more risk.

Bioreactors — whether adherent or suspension — are closed systems with reduced risk for contamination, since they require fewer seeding, transfection and harvest unit operations. They are also available in multiple sizes. The largest single-use bioreactors for suspension cell culture scale to 2000 L. 

An example of the industrialization of adherent cell culture has been accomplished in the form of the Pall iCELLis® disposable fixed-bed bioreactor system, the largest of which is 500 m2. This area translates roughly to a volume greater than 1000 L for a suspension bioreactor and is equivalent to 794 10-layer cell stacks or 5,882 roller bottles at 850 cm2 each  an order of magnitude increase in scale.

Timelines Drive Decision Making

The choice to manufacture viral vectors using an adherent or suspension cell culture system is based on several factors, though perhaps the most important driver is the timeline for the project. Regulatory authorities in many jurisdictions, including the United States, offer accelerated licensing approval pathways for cell and gene therapies, meaning that development and commercialization timelines can be shorter than those for traditional biologics.

The choice of culture system is driven by a number of factors, including the size of the product lot(s) needed in the clinic and marketplace, as well as the amount of time allotted for process development. Because adherent cell culture is familiar, and most viral vector processes are initially developed in flask-based systems, process development times, including scale-up in iCELLis® bioreactors, can be quicker. If scalability is more of a concern than a shorter timeline, then development of a robust suspension cell culture process might be preferred; however, it may require more time up front but it can, ultimately, pay dividends in terms of batch size. 

Other factors that influence the choice between adherent and suspension cell culture production include the disease target, the dose for each patient, the size of the patient population and the expected market penetration. The platform that can best support production of the desired quantity of viral vector is a primary driver. For some gene therapies, clinical and commercial production in plasticware may be sufficient, while other indications require production in bioreactors for commercial supply.

There are challenges from timeline and technical perspectives when switching to a new production platform after clinical trials have been performed in humans, particularly the need to demonstrate comparability of the product manufactured using the original and replacement process. Consequently, some drug companies elect to invest the time up front to develop processes and analytical methods that can be readily scaled to a commercially viable process.

Downstream Processing 

The upstream portion of viral vector manufacturing includes expanding the seed train, inoculating the terminal reactor and initiating production  steps that can take 3–5 weeks, and that are followed by 1–2 days required for downstream processing. The downstream portion is very important too, as it is essential to purify viral vectors from impurities to ensure that the final product is suitable for therapeutic use.

There are many variations in downstream processing, but a process generally begins with clarification of the harvested virus to remove cellular debris and other, larger impurities. The clarified harvest is then subjected to tangential flow filtration (TFF) to concentrate the viral vector particles and achieve buffer exchange. Chromatography is then performed to remove other remaining impurities, such as host-cell proteins, host-cell and plasmid DNA, etc. Ultrafiltration/diafiltration (UF/DF) via TFF is again performed to formulate the vector in the final buffer, and the formulated bulk vector is then subjected to sterile filtration and ultimately filling/finishing.

Manufacturers of hardware and consumables provide options to support most downstream unit operations for viral vector processing. For example, Pall’s Allegro MVP system with fully disposable flowpaths and single-use sensors for control and monitoring of key parameters can be used to run most downstream processes, including TFF, buffer preparation, pH adjustment, membrane chromatography, UF/DF and filling. It provides control of fully automated process sequences for optimal operations, greater consistency in product quality, reduced labor costs and reduction of operator errors.

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Because adherent cell culture is familiar, and most viral vector processes are initially developed in flask-based systems, process development times, including scale-up in iCELLis bioreactors, can be quicker.

Analytical Challenges

The complexity of viral vectors is much greater than traditional biologics. As a result, multiple orthogonal methods are employed to understand the physicochemical properties and quality of viral vector products. This multifaceted approach will continue unless breakthrough technologies are developed that enable the integration of the results from multiple analyses.

During process development, the “noise” in cell-based assays can also create challenges for the evaluation of process improvements. To overcome this difficulty, trending is performed to develop confidence that an improvement has been achieved. Fortunately, most methods used to determine physical properties, such as polymerase chain reaction (PCR) techniques, have greater accuracy.

Brammer Bio uses state-of-the-art technologies to verify the identity, strength and integrity of the genetic payload (e.g., vector genome), including digital droplet PCR8 for quantification, which is crucial for proper dosing. Next-generation sequencing techniques help with understanding the nucleic acid impurities, while high-performance liquid chromatography (HPLC) methods have replaced gel electrophoresis for purity analyses.

Evolving Technology

Until recently, the systems used for viral vector production have largely comprised tools and technologies designed for other applications, particularly mAb production.

Newer analytical techniques are providing a better understanding of the critical quality attributes of the vectors that are monitored during process development and manufacturing. New resins for affinity chromatography of certain vectors (such as POROS AAVX resin from Thermo Fisher Scientific) have been introduced for purification, and filtration technologies that take into account the specific challenges posed by viral vectors are also under development. 

Process controls tailored for viral vector production systems, which can have different cell culture profiles than mAbs and other recombinant proteins, are leading to more consistent processes and higher-quality products. Progress is also being achieved in developing better cell substrates for viral vector production.

Advances are being made on the drug product side as well, including formulation development, final product conditioning, fill/finish operations and labeling, storage and controlled transport   all of which present unique challenges for viral vectors. The goal is to ensure that the product reaches the patient with the greatest possible potency and safety. 

Outlook for the Future

Companies, including Pall, are working with viral vector manufacturers such as Brammer Bio to identify the needs for commercial viral vector production. They are actively investing in the development of new solutions and tools that are optimized specifically for viral vector upstream and downstream processing that will facilitate the manufacturing of these promising new treatments.

Brammer Bio has leveraged hardware technologies developed by Pall to provide enhanced services to its clients that require the scale-up of viral vector manufacturing processes. With a synergistic relationship, it is the patients who ultimately benefit from accelerating the development and commercialization of novel gene therapies.

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Scalable Adherent Production in the iCELLis Bioreactor

The PALL iCELLis® 500+ bioreactor is an automated, single-use, fixed-bed bioreactor that provides a large cell growth surface area within a small footprint. The compact fixed bed is filled with proprietary macrocarriers made of class VI polyester microfibers. Due to the cell-cell interactions within the 3D environment of the fixed bed, iCELLis bioreactors can be inoculated at very low densities (3,000 cells per cm2 or less), allowing for streamlined and simplified seed trains, fewer manual operations and reduced costs.

Evenly distributed media circulation is achieved by a built-in magnetic drive impeller, ensuring low shear stress and high cell viability. Media is pumped from the bottom through the packed bed and then cascades as a thin film down the outer walls, facilitating aeration and gas exchange. This unique waterfall oxygenation, together with gentle agitation and biomass immobilization, enables the compact iCELLis system to achieve and maintain high cell densities — achieving the productivity of much larger stirred-tank units. In addition, immobilization of the cells in the fixed bed combined with operation in perfusion/recirculation mode eliminates the need for centrifugation to harvest the cells, simplifying the downstream process.

Pall has investigated the production of various viral vectors using the iCELLis bioreactor and shown that it can enable the significant reduction of development timelines.2–4 Other researchers have also demonstrated the use of the iCELLis fixed-bed bioreactor technology for large-scale production of AV,5 AAV6 and LV7 vectors. 

The iCELLis 500 bioreactor is available in sizes ranging from 66 m2 to 500 m2, and with a choice of packed beds with lower and higher densities. The iCELLis Nano system (up to 4 m2) is also available for process development work and small-scale production. Moving from the small to larger bioreactors involves increasing the cross-sectional area of the fixed bed while maintaining a constant bed height. As a result, cell seeding and nutrient and oxygen delivery throughout the fixed bed are comparable. Pall has demonstrated that processes optimized in the iCELLis Nano scale directly to the iCELLis 500+ bioreactor with little additional work required.2

Figure 1

Figure 1. (A) Growth of insect cell line Sf9 in a Pall Allegro STR and cylindrical-vendor A* 200-L bioreactors. The cells were then co-infected with two baculovirus vectors to produce an rAAV5-GFP vector.  

(B) The ~3 μm diameter change in the cells is an indication of the progression of the infection in the Pall Allegro STR and cylindrical-vendor B* bioreactors. Clarified harvest: Pall Allegro STR 2.48×1011 vg/ml, cylindrical-vendor A 2.50×1011 vg/ml. *“A” and “B” signify two different cylindrical bioreactors. 

Acknowledgement

The Brammer Bio process development, analytical development and manufacturing teams performed the work presented in Figure 1.

Process controls tailored for viral vector production systems, which can have different cell culture profiles than mAbs and other recombinant proteins, are leading to more consistent processes and higher-quality products. Progress is also being achieved in developing better cell substrates for viral vector production.

References

  1. Kotin, R.G. and R.O. Snyder, “Manufacturing clinical grade recombinant adeno-associated virus using invertebrate cell lines.” Human Gene Therapy. 28:350-360 (2017).
  2. Knowles, S., J.C. Drugmand and J. Castillo. “Linear Scalability Of Virus Production in iCELLis® Single-Use, Fixed-Bed Bioreactors.” Pall Life Sciences. Sep. 2014. Web.
  3. Legmann, Rachel. “Industrialization of adenoviral vector production in an iCellis® 500 fixed bed bioreactor for the creation of autologous insulin producing liver cells for the treatment of diabetes: From bench to clinical scale.” PhDISCT Presentation. 2 Oct. 2016. Web.
  4. Legmann, Rachel. “Case study: Single-use platform for complete process development and scale-up of an Adenovirus” in Vaccine Technology VII, Amine Kamen, McGill University Tarit Mukhopadhyay, University College London Nathalie Garcon, Bioaster Charles Lutsch, Sanofi Pasteur Eds, ECI Symposium Series. 2018.
  5. Karhinen, Minna, et al. “Consistent Viral Vector Manufacturing for Phase III Using iCELLis(®) 500 Fixed-Bed Technology.” Vector and Cell Engineering/Manufacturing II. 24: S279 (2016).
  6. Powers AD, et al. “Development and Optimization of AAV hFIX Particles by Transient Transfection in an iCELLis(®) Fixed-Bed Bioreactor.” Hum. Gene Ther. Methods. 27:112–21 (2016).
  7. Valkama, A. J., al. “Optimization of lentiviral vector production for scale-up in fixed-bed bioreactor,” Gene Therapy. 25:39–46 (2018).
  8. Snyder, Richard, Diego Matayoshi, Susan D’costa, and Sushma Ogram. “Digital Droplet PCR for Viral Vector Analysis.” Pharma’s Almanac. 12 Mar. 2019.

Originally published on PharmasAlmanac.com on May 24, 2019