RESUMO
The recent FDA approval of several adeno-associated virus (AAV)-based gene therapies is driving demand for AAV production. One of the biggest AAV manufacturing challenges is removing "empty" capsids, which do not contain the gene of interest. Anion exchange chromatography has emerged as the leading solution for scalable full capsid enrichment. Here we develop a process for the baseline separation of empty and full AAV capsids using anion exchange membrane chromatography. This process development approach utilized AAV serotypes 8 and 9 and traverses initial screening of separation conditions up to manufacturing-scale processes. Process development of a two-step elution was performed via response surface DoE, exploring conductivity and the length of the first elution step. The results from response surfaces were used to construct statistical models of the process operating space. These models provide optimal conditions for recovery and purity, both of which can exceed 70 %. Model predictions were then validated at small scale prior to scale-up. We present the results from our scale-up purification and show that purity and yield are consistent with the results obtained from the response surface model.
Assuntos
Dependovirus , Dependovirus/genética , Dependovirus/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Humanos , Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/química , Proteínas do Capsídeo/isolamento & purificação , Proteínas do Capsídeo/análise , Células HEK293RESUMO
Adeno-associated viruses (AAVs) have emerged as promising tools for gene therapy due to their safety and efficacy in delivering therapeutic genes or gene editing sequences to various tissues and organs. AAV serotype 9 (AAV9), among AAV serotypes, stands out for its ability to efficiently target multiple tissues, thus holding significant potential for clinical applications. However, existing methods for purifying AAVs are cumbersome, expensive, and often yield inconsistent results. In this study, we explore a novel purification strategy utilizing Dynabeads™ CaptureSelect™ magnetic beads. The AAV9 magnetic beads capture AAV9 with high specificity and recovery between 70 and 90%, whereas the AAVX magnetic beads did not bind to the AAV9. Through continuous interaction with AAVs in solution, these beads offer enhanced clearance of genomic DNA and plasmids even in the absence of endonuclease. The beads could be regenerated at least eight times, and the used beads could be stored for up to six months and reused without a significant reduction in recovery. The potency of the AAV9-purified vectors in vivo was comparable to that of iodixanol purified vectors.
Assuntos
Dependovirus , Vetores Genéticos , Dependovirus/genética , Dependovirus/isolamento & purificação , Humanos , Vetores Genéticos/genética , Animais , Células HEK293 , Camundongos , Terapia Genética/métodosRESUMO
Purification of rAAV is a crucial unit operation of the AAV production process. It enables the capture of AAV and removal of contaminants such as host cell proteins, host cell DNA, and other cell culture-related impurities. Here we describe the purification of rAAV produced in insect cells Sf9/rBEV by immuno-affinity capture chromatography. The method is fully scale-amenable unlike other traditional purification methods based on ultracentrifugation. The method reported herein has two main steps: (1) the clarification of cell lysate by depth filtration and (2) the selective capture and single-step purification of AAV via immune-affinity chromatography. This purification method has been successfully implemented to purify the majority of wild-type AAV serotypes.
Assuntos
Cromatografia de Afinidade , Dependovirus , Dependovirus/genética , Dependovirus/isolamento & purificação , Animais , Cromatografia de Afinidade/métodos , Células Sf9 , Vetores Genéticos/genética , Humanos , Spodoptera/virologiaRESUMO
Data are presented demonstrating that absorbance detection can be used during high-speed sedimentation velocity analytical ultracentrifugation (hs-SV-AUC) experiments to characterize the size distribution of adeno-associated virus (AAV) drug products accurately. Advantages and limitations of being able to use this detector in this specific type of SV-AUC experiment are discussed.
Assuntos
Dependovirus , Ultracentrifugação , Dependovirus/genética , Dependovirus/isolamento & purificação , Ultracentrifugação/métodos , HumanosRESUMO
Mice infected with a recombinant adeno-associated virus carrying a replication-competent hepatitis B virus genome (rAAV-HBV) via the intravenous route establish a persistent HBV replication in hepatocytes and develop immune tolerance. They serve as models to evaluate antiviral immunity and to assess potential therapeutic approaches for chronic HBV infection. Combining selected HBV variants and different mouse genotypes allows for addressing a broad spectrum of research questions. This chapter describes the basic principles of the rAAV-HBV mouse model, rAAV-HBV production and purification methods, and finally, the in vivo application.
Assuntos
Dependovirus , Modelos Animais de Doenças , Vetores Genéticos , Vírus da Hepatite B , Replicação Viral , Animais , Dependovirus/genética , Dependovirus/isolamento & purificação , Vírus da Hepatite B/genética , Camundongos , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Humanos , Hepatite B Crônica/virologia , Hepatite B Crônica/imunologia , Hepatite B/virologia , Hepatite B/imunologiaRESUMO
Adeno-associated virus (AAV) vectors can efficiently transduce exogenous genes into various tissues in vivo. Owing to their convenience, high efficiency, long-term stable gene expression, and minimal side effects, AAV vectors have become one of the gold standards for investigating gene functions in vivo, especially in non-clinical studies. However, challenges persist in efficiently preparing a substantial quantity of high-quality AAV vectors. Commercial AAV vectors are typically associated with high costs. Further, in-laboratory production is hindered by the lack of specific laboratory equipment, such as ultracentrifuges. Therefore, a simple, quick, and scalable preparation method for AAV vectors is needed for proof-of-concept experiments. Herein, we present an optimized method for producing and purifying high-quality AAV serotype 9 (AAV9) vectors using standard laboratory equipment and chromatography. Using ceramic hydroxyapatite as a mixed-mode chromatography medium can markedly increase the quality of purified AAV vectors. Basic Protocols and optional methods for evaluating purified AAV vectors are also described. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of AAV9 vectors in 293EB cells Basic Protocol 2: Concentration and buffer exchange of AAV9 vectors from 293EB cell culture supernatants using tangential flow filtration Basic Protocol 3: Purification of AAV9 vectors from TFF samples using ceramic hydroxyapatite chromatography Basic Protocol 4: Analysis of the purified AAV9 vectors.
Assuntos
Cerâmica , Dependovirus , Durapatita , Vetores Genéticos , Sorogrupo , Dependovirus/genética , Dependovirus/isolamento & purificação , Vetores Genéticos/isolamento & purificação , Vetores Genéticos/genética , Humanos , Cerâmica/química , Durapatita/química , Cromatografia/métodos , Células HEK293RESUMO
Here, we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium using orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI)-mediated transfection of a three-plasmid system and is specified for production in milliliter-to-liter scales. After PEI and plasmid DNA (pDNA) complex formation, the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 7-day batch process, cell cultures are further processed using a set of methods for cell lysis and vector recovery. Methods for the purification of viral particles are described, including immunoaffinity and anion-exchange chromatography, ultrafiltration, as well as digital PCR to quantify the concentration of vector particles.
Assuntos
Dependovirus , Vetores Genéticos , Transfecção , Humanos , Dependovirus/genética , Dependovirus/isolamento & purificação , Células HEK293 , Vetores Genéticos/genética , Vetores Genéticos/isolamento & purificação , Transfecção/métodos , Plasmídeos/genética , Plasmídeos/isolamento & purificação , Polietilenoimina/química , Reatores Biológicos , Cromatografia por Troca Iônica/métodos , Vírion/genética , Vírion/isolamento & purificaçãoRESUMO
The field of recombinant adeno-associated virus (rAAV) gene therapy has attracted increasing attention over decades. Within the ongoing challenges of rAAV manufacturing, the co-production of impurities, such as empty and partial capsids containing no or truncated transgenes, poses a significant challenge. Due to their potential impact on drug efficacy and clinical safety, it is imperative to conduct comprehensive monitoring and characterization of these impurities prior to the release of the final gene therapy product. Nevertheless, existing analytical techniques encounter notable limitations, encompassing low throughput, long turnaround times, high sample consumption, and/or complicated data analysis. Chromatography-based analytical methods are recognized for their current Good Manufacturing Practice (cGMP) alignment, high repeatability, reproducibility, low limit of detection, and rapid turnaround times. Despite these advantages, current anion exchange high pressure liquid chromatography (AEX-HPLC) methods struggle with baseline separation of partial capsids from full and empty capsids, resulting in inaccurate full-to-empty capsid ratio, as partial capsids are obscured within peaks corresponding to empty and full capsids. In this study, we present a unique analytical AEX method designed to characterize not only empty and full capsids but also partial capsids. This method utilizes continuous N-Rich chromatography with recycling between two identical AEX columns for the accumulation and isolation of partial capsids. The development process is comprehensively discussed, covering the preparation of reference materials representing full (rAAV-LacZ), partial (rAAV-GFP), and empty (rAAV-empty) capsids, N-rich method development, fraction analysis, determination of fluorescence response factors between capsid variants, and validation through comparison with other comparative techniques.
Assuntos
Capsídeo , Dependovirus , Dependovirus/genética , Dependovirus/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Capsídeo/química , Cromatografia Líquida de Alta Pressão/métodos , Reprodutibilidade dos TestesRESUMO
Ultracentrifugation is an attractive method for separating full and empty capsids, exploiting their density difference. Changes of the serotype/capsid, density of loading material, or the genetic information contained in the adeno-associated viruses (AAVs) require the adaptation of the harvesting parameters and the density gradient loaded onto the centrifuge. To streamline these adaptations, a mathematical model could support the design and testing of operating conditions.Here, hybrid models, which combine empirical functions with artificial neural networks, are proposed to describe the separation of full and empty capsids as a function of material and operational parameters, i.e., the harvest model. In addition, critical quality attributes are estimated by a quality model which is operating on top of the harvest model. The performance of these models was evaluated using test data and two additional blind runs. Also, a "what-if" analysis was conducted to investigate whether the models' predictions align with expectations.It is concluded that the models are sufficiently accurate to support the design of operating conditions, though the accuracy and applicability of the models can further be increased by training them on more specific data with higher variability.
Assuntos
Dependovirus , Ultracentrifugação , Dependovirus/genética , Dependovirus/isolamento & purificação , Ultracentrifugação/métodos , Vírion/isolamento & purificação , Vírion/química , Redes Neurais de ComputaçãoRESUMO
Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs. This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.