Accelerating The Development of Viral Vector Manufacturing Processes

INDUSTRY LEADER INSIGHT: Viral Vector Manufacturing

As cell and gene therapies have the potential to rapidly advance through clinical trials to commercialization, there is increasing demand for practical manufacturing solutions for viral vectors that can be readily optimized and scaled. Through ongoing efforts, Brammer Bio and Pall Biotech are each developing and implementing state-of-the-art solutions designed to accelerate process development and scale-up for 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.


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.



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. 


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.


  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.

Richard Snyder, Ph.D.

Dr. Snyder was the founder of Florida Biologix, which was spun out of the University of Florida in 2015 and merged to create Brammer Bio in 2016. Dr. Snyder has been investigating virus biology, vector development, cGMP manufacturing and analytical technologies, and viral vector–mediated gene transfer for over 32 years. Dr. Snyder received his doctoral degree in microbiology from the State University of New York at Stony Brook and obtained his BA in biology from Washington University in St. Louis.