Overcoming Bottlenecks in AAV Manufacturing for Gene Therapy

Smith, Jacob; Grieger, Josh; Samulski, R. Jude

doi:10.18609/cgti.2018.083

Cited by 11 publications

(8 citation statements)

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“…Chromatography matrix References Affinity A20 Grimm et al (1998) Heparin (POROS HE) Anderson et al (2000) Mucin Auricchio et al (2001) AVB Sepharose HP Dismuke and Kotin (2017), Gu et al (2018), Hebben (2018), Nass et al (2018), J. Smith, Grieger, and Samulski (2018), R. H. Smith, Levy, and Kotin (2009), Terova et al (2018), Toueille (2018) POROS AAVX Hebben (2018), Terova et al (2018), Toueille (2018) Anion exchange POROS 50 HQ Urabe et al (2006) Q sepharose FF Tomono et al (2018) Cation exchange Fractogel® EMD SO 3 − Chahal, Aucoin, and Kamen (2007) Mustang S Davidoff et al (2004) POROS 50 HS Heldt 2019Hydrophobic interaction CIMmultus OH Leskovec et al (2018) and full capsid quantity due the difference in DNA present in full capsids based on the predictable manner in which empty capsids reduce the absorbance A 260 /A 280 ratio (Sommer et al, 2003). Charge detection mass spectrometry can be used by measuring the mass charge ratio and the individual ion charge (Pierson, Keifer, Asokan, & Jarrold, 2016).…”

Section: Capture Methodsmentioning

confidence: 99%

Moving from the bench towards a large scale, industrial platform process for adeno‐associated viral vector purification

Adams

Bak

Tustian

2020

Biotech & Bioengineering

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In recent years, there has been a strong interest in the development and production of gene therapy products, especially those utilizing adeno‐associated virus (AAV) particles. This is evident with the growing number of clinical successes and agency approvals for AAV therapeutics. Due to this increased investment in this technology, a need exists for scalable commercial production methods to ensure adequate product supply as research in AAV shifts from bench‐scale development to clinical production. The purpose of this review is to summarize current scalable purification techniques that can be employed during the commercial manufacturing of AAV as well as highlight certain development considerations, such as adventitious agent removal and process development using the principals of quality by design.

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Section: Capture Methodsmentioning

confidence: 99%

Moving from the bench towards a large scale, industrial platform process for adeno‐associated viral vector purification

Adams

Bak

Tustian

2020

Biotech & Bioengineering

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“…Thus, the integration pattern and its potential implication for oncogenesis due to rAAV vectors differ from that of WT-AAV. Of the four currently available distinct rAAV production platforms suitable for scaling up vector production, 25 two have been most commonly used for hemophilia gene therapy vectors. Hemophilia rAAV vectors to date have been produced via either transfection of mammalian cells with naked plasmid DNA or introduction of baculovirus expression vectors into Spodoptera frugiperda (Sf9) insect cells followed by cell lysis and purification via cesium chloride (CsCl) gradient sedimentation or ion exchange chromatography.…”

Section: Aav-based Gene Therapy: the Factsmentioning

confidence: 99%

Guest Editor: G. Castaman GENE THERAPY FOR HEMOPHILIA: FACTS AND QUANDARIES IN THE 21ST CENTURY

Arruda

Doshi

2020

Mediterr J Hematol Infect Dis

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Therapy for hemophilia has evolved in the last 40 years from plasma-based concentrates to recombinant proteins and, more recently, to non-factor therapeutics. Along this same timeline, research in adeno-associated virus (AAV) based gene therapy vectors has provided the framework for successful early phase clinical trials initially for hemophilia B (HB) and now for hemophilia A. Successive lessons learned from these early pivotal HB trials have paved the way for current advanced phase trials. Nevertheless, questions linger regarding 1) the optimal balance of vector dose to transgene expression, 2) amount and durability of transgene expression required, and 3) long-term safety. Some trials have demonstrated unique findings not seen previously regarding transient elevation of liver enzymes, immunogenicity of the vector capsid, and loss of transgene expression. This review will provide an update on the clinical AAV gene therapy trials in hemophilia and address the questions above. A thoughtful and rationally approached expansion of gene therapy to the clinics would certainly be a welcome addition to the arsenal of options for hemophilia therapy. Further, the global impact of gene therapy could be vastly improved by expanding eligibility to different patient populations and to developing nations. With the advances made to date, it is possible to envision a shift from the early modest goal of simply increasing life expectancy to a significant improvement in quality of life by reduction in spontaneous bleeding episodes and disease complications.

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“…However, the quantity of AAV required for such applications varies from 1 × 10 13 (e.g., Leber’s congenital amaurosis) to 5 × 10 20 viral genomes (e.g., Duchenne muscular dystrophy), placing substantial pressure on manufacturing processes ( Koilkonda et al, 2014 ; Crudele and Chamberlain, 2019 ). Despite the several technologies available, the upstream processing of AAV is still one of the main bottlenecks of clinical-grade AAV manufacturing ( Smith et al, 2018 ) with specific production titers and vector quality (i.e., % full particles) being two of the most challenging parameters to control and/or optimize ( Merten, 2016 ). The implementation of process intensification strategies has already been demonstrated to overcome these challenges with successful case studies for monoclonal antibodies ( Chen et al, 2018 ; Xu et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

AAV process intensification by perfusion bioreaction and integrated clarification

Mendes

Fernandes

Pineda

et al. 2022

Front. Bioeng. Biotechnol.

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Adeno-associated viruses (AAVs) demand for clinical trials and approved therapeutic applications is increasing due to this vector’s overall success and potential. The high doses associated with administration strategies challenges bioprocess engineers to develop more efficient technologies and innovative strategies capable of increasing volumetric productivity. In this study, alternating tangential flow (ATF) and Tangential Flow Depth filtration (TFDF) techniques were compared as to their potential for 1) implementing a high-cell-density perfusion process to produce AAV8 using mammalian HEK293 cells and transient transfection, and 2) integrating AAV harvest and clarification units into a single step. On the first topic, the results obtained demonstrate that AAV expression improves with a medium exchange strategy. This was evidenced firstly in the small-scale perfusion-mocking study and later verified in the 2 L bioreactor operated in perfusion mode. Fine-tuning the shear rate in ATF and TFDF proved instrumental in maintaining high cell viabilities and, most importantly, enhancing AAV-specific titers (7.6 × 104 VG/cell), i.e., up to 4-fold compared to non-optimized perfusion cultures and 2-fold compared with batch operation mode. Regarding the second objective, TFDF enabled the highest recovery yields during perfusion-based continuous harvest of extracellular virus and lysate clarification. This study demonstrates that ATF and TFDF techniques have the potential to support the production and continuous harvest of AAV, and enable an integrated clarification procedure, contributing to the simplification of operations and improving manufacturing efficiency.

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Overcoming Bottlenecks in AAV Manufacturing for Gene Therapy

Cited by 11 publications

References 0 publications

Moving from the bench towards a large scale, industrial platform process for adeno‐associated viral vector purification

Moving from the bench towards a large scale, industrial platform process for adeno‐associated viral vector purification

Guest Editor: G. Castaman GENE THERAPY FOR HEMOPHILIA: FACTS AND QUANDARIES IN THE 21ST CENTURY

AAV process intensification by perfusion bioreaction and integrated clarification

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