Optimization of AAV Process Development: Transfection Matters

Summary 

With the increased number of therapeutic rAAV candidates entering the clinical trial phase, there is a growing demand for innovative technologies to enhance process development and facilitate manufacturing scale-up for future commercialization. To address this need, Sartorius Polyplus has collaborated closely with viral vector manufacturers to develop transfection reagents specifically for large scale manufacturing. One such reagent is FectoVIR^®-AAV, which aims to improve rAAV manufacturing processes by boosting productivity, bringing flexibility and facilitating scalability. Here, we present data from Allergan Biologics’ evaluation of FectoVIR^®-AAV compared to their current AAV production platform. Analysis of physical titers revealed a 3-fold increase in both viral particles (VP) and viral genome (VG) per ml of cell culture when using FectoVIR®-AAV transfection reagent compared to PEIpro^®.  

rAAVs at Center Stage for Gene Therapy 

Adeno-associated viruses (AAVs) are recombinant viral vectors used to deliver corrective gene therapies into target cells, offering a promising approach to address a wide spectrum of monogenic diseases, including blood disorders, neurological and ocular diseases. Initially discovered as a contaminant in adenovirus preparation [1], AAVs have been propelled to the forefront due to their unique biological and physical properties. AAVs are small, 25 nm, non-enveloped and replication-defective viruses that have not been linked to any known human diseases [2]. Unlike wild-type AAVs, recombinant AAVs (rAAVs) are genetically modified to remove all viral coding sequences (rep and cap) from their 4.8 kb genome, thereby allowing the packaging of a transgene expression cassette of up to 4.4 kb in size [3]. Without its viral coding sequences, rAAVs function is restricted to being a protein-based nanoparticle that carries a DNA cargo into the nucleus of cells. In the absence of the replication gene (rep), the DNA cargo, once delivered into the nucleus, does not undergo site-specific integration into the genome of cells and instead persists as episomal DNA, as long as cells do not actively divide [4]. This latency as episomal DNA, which permits long-term expression in cells, largely contributes to the excellent safety profile and ensuing popularity of AAV-based gene therapy strategies. 

The popularity of AAV-based gene therapy is also attributable to the existence of numerous AAV variants with tissue and cell specific tropisms. With the identification of a human cell line that could not be transduced by the first characterized AAV-2 [5], research on the transduction mechanism of AAVs led to identification of a combination of cellular receptors and co-receptors for AAV-2 to gain entry into cells. This led to the identification of 13 naturally occurring human and primate AAV serotypes (AAV1-AAV13) and more than 100 AAV variants across animal species [6]. AAV serotypes can be chosen for their tropism, with the ability to preferentially transduce a specific cell or tissue type, or for their ability to have a broad tropism. For example, AAV2, known for its broad tropism, has been approved as a viral vector for the treatment of an inherited form of retinal disease (Luxturna^®), while AAV serotypes with a more specific tissue tropism have been approved for the treatment of inherited lipoprotein lipase deficiency (AAV1; Glybera^®) and inherited spinal muscular atrophy (AAV9; Zolgensma^®). 

Addressing rAAV Manufacturing Bottlenecks: Productivity and Scalability 

With the increased number of therapeutic rAAV applications reaching clinical trial, and the high doses of rAAV often being administered, production of rAAVs in sufficient amounts within acceptable cost limitations has become critical. rAAVs are mainly produced in human HEK-293 cells, which requires the transfection of these cells with up to three plasmids containing elements needed for AAV viral vector assembly: a plasmid carrying the transgene expression cassette, a plasmid carrying the rep/cap genes and a third plasmid containing helper genes provided by adenovirus or herpes virus [4]. Co-delivery of these plasmids in cells is crucial to producing functional viral particles. Therefore, the transfection method is critical to ensure efficient co-delivery, in as many cells as possible and, importantly, in a reproducible manner to ensure robust production yields irrespective of the manufacturing scale. 

Viral vector production mainly relies on suspension cell systems to achieve higher production rates per batch and flexible, scalable manufacturing processes [7]. Therefore, there is a demand for a transfection reagent that can support scale-up of rAAV manufacturing by fulfilling the following criteria: (i) improve rAAV viral vector yields, (ii) ensure process scalability for large scale manufacturing, (iii) enhance batch-to-batch reproducibility, and (iv) importantly, comply with quality and regulatory requirements for GMP manufacturing and commercialization. Sartorius Polyplus’ innovative transfection reagent, FectoVIR®-AAV, addresses many of these requirements [8]. It is a novel class of animal-free transfection reagent that is specifically developed for large scale transfection in suspension and adherent cell systems. Due to its unique physico-chemical properties, FectoVIR®-AAV can improve productivity at large scale, decrease batch-to-batch variability and simplify scale-up with a transfection protocol that addresses time and volume constraints [7]. 

Case Study: Allergan Biologics’ Evaluation of FectoVIR®-AAV 

Allergan Biologics, an Abbvie company, is a Centre of Excellence for Biologics R&D, with a focus on the development and manufacture of gene therapy products. Due to their strong interest in the investigation of innovative technologies, Allergan Biologics  tested FectoVIR®-AAV against their existing AAV production platform . The comparison of FectoVIR®-AAV  with another transfection reagent, referred to subsequently as the “market alternative”, was  conducted side-by side with PEIpro®. PEIpro® transfection reagent (Sartorius Polyplus) is optimized for small to large-scale transfection of both adherent and suspension HEK-293 cells, used in the manufacturing of viral vectors such as lentivirus, AAVs and virus-like particles. Since the availability of GMP (Good Manufacturing Practices)-compliant PEIpro® in 2018, PEIpro® has become the first PEI-based transfection reagent that can support viral manufacturers from process development to commercialization. 

For viral manufacturers, the current challenge is to optimize AAV production platforms by achieving high production yields in HEK-293 suspension cell culture systems. In comparison to PEIpro® and the market alternative, FectoVIR®-AAV improved AAV productivity when tested against Allergan Biologic’s rAAV production platform. AAV production yields were assessed by measuring viral particles (VP) and genome copies (VG) number per milliliter of harvested cell culture. FectoVIR®-AAV led to approximately a 3-fold increase in physical titers as measured in VP/ml and VG/ml compared to PEIpro®, and was also superior to the market alternative (Figure 1 & Figure 2). In addition, cell viability at the time of harvest was measured following transfection with PEIpro®, FectoVIR®-AAV and market alternative. In all cases, cell viability was above 80% at harvest (Figure 3).  High viability at harvest has the potential to positively impact downstream processing due to an improved impurity profile. 

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Meeting Compliance with Quality Requirements 

In addition to improving AAV production yields, a transfection reagent must also fulfill regulatory requirements, as increasing manufacturing capacity demands that all materials used in the production of AAV gene therapy vectors meet stringent quality and traceability standards. Sartorius Polyplus is a pioneer in the manufacturing of GMP transfection reagents for gene therapy, with the launch of PEIpro®-GMP in 2018 and FectoVIR®-AAV GMP in 2023, accompanied by a residual test to support the clearance of process-related impurities of the transfection reagent throughout the AAV manufacturing process. 

References 

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Affiliations 

• Kelsey Wosnitzka| Industrial Placement Student, Process Sciences, Allergan Biologics (an Abbvie company) 

• Shandel Pariag | Senior Scientist, Allergan Biologics(an Abbvie company) 

• Hélène Trottin | Senior Technical Specialist, Process Sciences, Allergan Biologics(an Abbvie company) 

• Alengo Nyamay’antu | Segment Marketing Manager, Sartorius Polyplus 

• Mathieu Porte | R&D Manager Bioproduction, Sartorius Polyplus

• Malik Hellal | Senior Scientist Chemistry, Sartorius Polyplus

• Patrick Erbacher | CSO ,Sartorius Polyplus

Authorship & Conflict of Interest 

Contributions: All named authors take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published. 

Acknowledgements: None. 

Disclosure and potential conflicts of interest: The authors declare that they have no conflicts of interest. 

Funding declaration: The authors received no financial support for the research, authorship and/or publication of this article. 

Article & copyright information 

Copyright: Published by Cell and Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0 which allows anyone to copy, distribute, and transmit the article provided it is properly attributed in the manner specified below. No commercial use without permission. 

Attribution: Copyright © 2021 Wosnitzka K, Pariag S, Trottin H, Nyamay’antu A, Porte M, Hellal M, Erbacher P. Published by Cell and Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0.