Production, Processing, and Characterization of Synthetic AAV Gene Therapy Vectors

Abstract

Over the last two decades, gene therapy vectors based on wild‐type Adeno‐associated viruses (AAV) are safe and efficacious in numerous clinical trials and are translated into three approved gene therapy products. Concomitantly, a large body of preclinical work has illustrated the power and potential of engineered synthetic AAV capsids that often excel in terms of an organ or cell specificity, the efficiency of in vitro or in vivo gene transfer, and/or reactivity with anti‐AAV immune responses. In turn, this has created a demand for new, scalable, easy‐to‐implement, and plug‐and‐play platform processes that are compatible with the rapidly increasing range of AAV capsid variants. Here, the focus is on recent advances in methodologies for downstream processing and characterization of natural or synthetic AAV vectors, comprising different chromatography techniques and thermostability measurements. To illustrate the breadth of this portfolio, two chimeric capsids are used as representative examples that are derived through forward‐ or backwards‐directed molecular evolution, namely, AAV‐DJ and Anc80. Collectively, this ever‐expanding arsenal of technologies promises to facilitate the development of the next AAV vector generation derived from synthetic capsids and to accelerate their manufacturing, and to thus boost the field of human gene therapy.

Single-Use Capture Purification of Adeno-Associated Viral Gene Transfer Vectors by Membrane-Based Steric Exclusion Chromatography

“…9,10,13,14 Indeed, other methods have been developed and applied for AAV purification that comply with current good manufacturing practice and that are typically based on chromatography techniques. 15 One notable variant is ion exchange chromatography (IEX) that can separate full and empty AAV capsids and has already been used for purification of several AAV serotypes comprising AAV2, AAV4, AAV5, and AAV8. [16][17][18][19] However, the IEX process is not universally applicable but rather needs to be adapted and optimized for each AAV capsid variant.…”

Viral vector‐based gene therapies in the clinic

“…Viral vector‐based gene therapies face many challenges that can affect their implementation and adoption into standard clinical care; indeed, these challenges have been extensively reviewed in previous articles. 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 A main challenge is the reliance on animal models that inaccurately predict transduction efficiency in humans; one effort in this area is the development of humanized mouse models or animal models that use tissue grafts from humans to better predict potential efficacy and success of viral vector‐based gene therapies in the clinic. 42 Closely connected to efficacy in humans, immune response from viral vectors presents a significant hurdle to the translation of viral vector‐based gene therapies.…”

“…Approaches to mitigate immune responses against viral vectors include introduction of novel vectors with tissue‐specific tropism, 104 genetically engineering the capsid through directed evolution, 42 and altering the route of administration, 105 to name a few currently emerging approaches. 106 , 107 , 108 There also exists considerable manufacturing challenges, which have been previously and extensively reviewed 95 , 96 , 97 , 101 , 102 , 103 ; importantly, these limitations with manufacturing and productions can contribute to the high costs of many viral vector‐based gene therapies, especially those for rare diseases. 98 To address these production and manufacturing challenges, emerging efforts include transition from transfected producer cells to stable producer cell lines, 95 development of processes that enable suspension culture (as opposed to adherent‐culture) of producer cell lines, 96 development of serotype‐independent purification processes for viral vectors, 109 and new methodologies to identify contaminating proteins or bacteria of vector stocks, 102 to name a few.…”

Biomanufacturing of Gene Therapy Vectors

“…[ 7 ] review recent progress in the purification of lentiviral vectors. El Andari and Grimm‘s [ 8 ] paper present in their review methods for the purification of different serotypes of AAV vectors with special emphasis on synthetic AAV gene therapy vectors (AAV‐DJ and ancAAV). Furthermore, Dickerson et al.…”

“…This requires, in particular, viral vectors destined for in vivo use, such as AAV vectors, the availability of efficient monitoring methods during process development and proper characterization, and robust quantification methods in the phase of routine manufacturing to cope with regulatory demands. In the context of process development, papers by Wright, [ 10 ] Lecomte et al., [ 11 ] and El Andari and Grimm [ 8 ] present different aspects of analytical methods required for the deep characterization of rAAV vectors for R&D as well as for process developmental purposes, including, for instance, the development of an improved method for high throughput sequencing to identify and quantify DNA species in recombinant AAV batches, [ 11 ] mass spectrometry‐based methods to characterize AAV capsid protein integrity, [ 8 ] vp1,2,3 stoichiometry, [ 8 ] deamidation [ 10,12 ] and oxidation [ 10,13 ] /phosphorylation [ 13 ] or differential scanning fluorimetry to measure AAV capsid thermostability. [ 8 ] Furthermore, the paper by Wright [ 10 ] reviews quality control methods for routine testing of AAV vectors.…”