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Cell therapies have gained prominence as a promising therapeutic modality for treating a range of diseases. Despite the recent clinical successes of cell therapy products, very few formal training programs exist for cell therapy manufacturing. To meet the demand for a well-trained workforce, we assembled a team of university researchers and industry professionals to develop an online course on the principles and practice of cell therapy manufacturing. The course covers the basic cell and systems physiology underlying cell therapy products, in addition to explaining end-to-end manufacturing from cell acquisition through to patient treatment, industrialization, and regulatory processes. So far, over 10,000 learners have enrolled in the course, and over 90% of respondents to the course exit survey indicated that they were 'very likely' or 'likely' to recommend the course to a peer. In this paper, we discuss our experience in the collaborative design and implementation of the online course, as well as lessons learned from quantitative and qualitative student feedback. We believe that this course can serve as a model for how academia and industry can collaborate to create innovative, scalable training programs to meet the demands of the modern biotechnology workforce.
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The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) collected historical data from 20 biopharmaceutical industry members on their experience with the in vivo adventitious virus test, the in vitro virus test, and the use of next generation sequencing (NGS) for viral safety. Over the past 20 years, only three positive in vivo adventitious virus test results were reported, and all were also detected in another concurrent assay. In more than three cases, data collected as a part of this study also found that the in vivo adventitious virus test had given a negative result for a sample that was later found to contain virus. Additionally, the in vivo adventitious virus test had experienced at least 21 false positives and had to be repeated an additional 21 times all while using more than 84,000 animals. These data support the consideration and need for alternative broad spectrum viral detection tests that are faster, more sensitive, more accurate, more specific, and more humane. NGS is one technology that may meet this need. Eighty one percent of survey respondents are either already actively using or exploring the use of NGS for viral safety. The risks and challenges of replacing in vivo adventitious virus testing with NGS are discussed. It is proposed to update the overall virus safety program for new biopharmaceutical products by replacing in vivo adventitious virus testing approaches with modern methodologies, such as NGS, that maintain or even improve the final safety of the product.
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Produtos Biológicos , Vírus , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Vírus/genética , Contaminação de Medicamentos/prevenção & controleRESUMO
Quantifying the composition of viral vectors used in vaccine development and gene therapy is critical for assessing their functionality. Adeno-associated virus (AAV) vectors, which are the most widely used viral vectors for in vivo gene therapy, are typically characterized using PCR, ELISA, and analytical ultracentrifugation which require laborious protocols or hours of turnaround time. Emerging methods such as charge-detection mass spectroscopy, static light scattering, and mass photometry offer turnaround times of minutes for measuring AAV mass using optical or charge properties of AAV. Here, we demonstrate an orthogonal method where suspended nanomechanical resonators (SNR) are used to directly measure both AAV mass and aggregation from a few microliters of sample within minutes. We achieve a precision near 10 zeptograms which corresponds to 1% of the genome holding capacity of the AAV capsid. Our results show the potential of our method for providing real-time quality control of viral vectors during biomanufacturing.
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Dependovirus , Vetores Genéticos , Capsídeo , DNA , Dependovirus/genética , Vetores Genéticos/genéticaRESUMO
In recent years, high temperature short time (HTST) treatment technology has been increasingly adopted for medium treatment to mitigate the potential risk of viral contamination in mammalian cell culture GMP manufacturing facilities. Mouse minute virus (MMV), also called minute virus of mice (MVM), implicated in multiple viral contamination events is commonly used as a relevant model virus to assess the effectiveness of HTST treatment of cell culture media. However, results from different studies vary broadly in inactivation kinetics as well as log reduction factors (LRFs) achieved under given treatment conditions. To determine whether the reported discrepancies stemmed from differences in MMV strains, laboratory-scale HTST devices, medium matrices, and/or experimental designs, we have taken a collaborative approach to systematically assess the effectiveness of HTST treatment for MMV inactivation. This effort was conceptualized based on a media treatment gap analysis conducted by the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) under the MIT Center for Biomedical Innovation (CBI). Specifically, two different MMV strains were used to evaluate the effectiveness of HTST at various treatment conditions with regard to exposure temperature and hold time duration by two independent laboratories within two different companies. To minimize experimental variations, the two sites used the same batches of MMV stocks, the same commercially purchased medium, and the same model of thermocyclers as the laboratory-scale HTST device. The two independent laboratories yielded similar MMV inactivation kinetics and comparable LRF. No significant differences were observed between the two MMV strains evaluated, suggesting that the variations from prior studies were likely due to differences in equipment, medium matrices, or other factors. The data presented here indicate that MMV inactivation by HTST treatment obeys first-order kinetics and can be mathematically modeled using an Arrhenius equation. The model-based extrapolation provides a quantitative estimate of MMV inactivation by the current industry standard HTST condition (102°C for a hold time of 10 s) used for medium treatment. Finally, based on the data from the current study and the industry experience, it is recommended that any alternative virus barrier technologies adopted for medium treatment should provide a clearance of at least 3.0 LRF based on a worst-case model virus to effectively mitigate potential risks of viral contamination.
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Temperatura Alta , Vírus Miúdo do Camundongo/química , Inativação de Vírus , Animais , Linhagem Celular Transformada , Humanos , Camundongos , Fatores de TempoRESUMO
Therapeutic proteins (TPs) are critical in modern medicine, yet shortage of TPs in disaster situations and remote areas remains a worldwide challenge. Manufacturing and real-time release of TPs on demand at the point-of-care is considered the key to this issue, which requires reliable and rapid analytics techniques for quality assurance. Herein we report a microfluidic platform that could be implemented in-line and at the point-of-care for real-time decision-making about the quality of a TP. The in vivo efficacy and duration of efficacy of TPs were assessed by the equilibrium and kinetics of TP and TP receptor (TPR) binding, using electrokinetic concentration (EC) and molecular charge modulation (MCM). EC can simultaneously concentrate and separate bound and unbound species in an assay based on electrical mobility, allowing for the quantification of binding. MCM enables the application of EC to arbitrary TPs by enhancing the mobility differences between TPs, TPRs, and TP-TPR complexes. This technology is homogeneous and overcomes many practical challenges of conventional heterogeneous assays. We developed various formats of assays for equilibrium and kinetic analysis and rapid determination of degradation of TPs, obtaining results comparable to state-of-the-art technologies with significantly less time (<1 h) and simpler setup. Finally, we demonstrated that the results of MCM-EC based assays correlated well with those from mass spectrometry and cell-based assay, which are the industrial standards for quality testing of TPs.
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Cromatografia Capilar Eletrocinética Micelar , Fator Estimulador de Colônias de Granulócitos/análise , Hormônio do Crescimento Humano/análise , Interferon-alfa/análise , Técnicas Analíticas Microfluídicas , Humanos , Interferon alfa-2 , Cinética , Proteínas Recombinantes/análiseRESUMO
Protein A is often used for the purification and detection of antibodies such as immunoglobulin G (IgG) because of its quadrivalent domains that bind to the Fc region of these macromolecules. However, the kinetics and thermodynamics of the binding to many sensor surfaces have eluded mechanistic description due to complexities associated with multivalent interactions. In this work, we use a near-infrared (nIR) fluorescent single-walled carbon nanotube sensor array to obtain the kinetics of IgG binding to protein A, immobilized using a chelated Cu(2+)/His-tag chemistry to hydrogel dispersed sensors. A bivalent binding mechanism is able to describe the concentration dependence of the effective dissociation constant, KD,eff, which varies from 100 pM to 1 µM for IgG concentrations from 1 ng mL(-1) to 100 µg mL(-1), respectively. The mechanism is shown to describe the unusual concentration-dependent scaling demonstrated by other sensor platforms in the literature as well, and a comparison is made between resulting parameters. For comparison, we contrast IgG binding with that of human growth hormone (hGH) to its receptor (hGH-R) which displays an invariant dissociation constant at KD = 9 µM. These results should aid in the use of protein A and other recognition elements in a variety of sensor types.
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Técnicas de Química Analítica/instrumentação , Técnicas de Química Analítica/métodos , Proteínas Imobilizadas/química , Imunoglobulina G/química , Análise em Microsséries , Proteína Estafilocócica A/química , Fluorescência , Hormônio do Crescimento Humano/química , Humanos , Nanotubos de Carbono/química , Ligação Proteica , Propriedades de SuperfícieRESUMO
A class of peptides from the bombolitin family, not previously identified for nitroaromatic recognition, allows near-infrared fluorescent single-walled carbon nanotubes to transduce specific changes in their conformation. In response to the binding of specific nitroaromatic species, such peptide-nanotube complexes form a virtual "chaperone sensor," which reports modulation of the peptide secondary structure via changes in single-walled carbon nanotubes, near-infrared photoluminescence. A split-channel microscope constructed to image quantized spectral wavelength shifts in real time, in response to nitroaromatic adsorption, results in the first single-nanotube imaging of solvatochromic events. The described indirect detection mechanism, as well as an additional exciton quenching-based optical nitroaromatic detection method, illustrate that functionalization of the carbon nanotube surface can result in completely unique sites for recognition, resolvable at the single-molecule level.
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Hidrocarbonetos Aromáticos/análise , Chaperonas Moleculares/análise , Nanotubos de Carbono/química , Nitrocompostos/análise , Peptídeos/química , Adsorção , Fluorescência , Microscopia/instrumentação , Estrutura Secundária de ProteínaRESUMO
Second near-infrared (NIR) window light (950-1400 nm) is attractive for in vivo fluorescence imaging due to its deep penetration depth in tissues and low tissue autofluorescence. Here we show genetically engineered multifunctional M13 phage can assemble fluorescent single-walled carbon nanotubes (SWNTs) and ligands for targeted fluorescence imaging of tumors. M13-SWNT probe is detectable in deep tissues even at a low dosage of 2 µg/mL and up to 2.5 cm in tissue-like phantoms. Moreover, targeted probes show specific and up to 4-fold improved uptake in prostate specific membrane antigen positive prostate tumors compared to control nontargeted probes. This M13 phage-based second NIR window fluorescence imaging probe has great potential for specific detection and therapy monitoring of hard-to-detect areas.
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Bacteriófago M13/fisiologia , Microscopia de Fluorescência/métodos , Técnicas de Sonda Molecular , Nanotubos de Carbono/análise , Neoplasias da Próstata/patologia , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias da Próstata/virologiaRESUMO
Current manufacturing processes for recombinant adeno-associated viruses (rAAVs) have less-than-desired yields and produce significant amounts of empty capsids. The increasing demand and the high cost of goods for rAAV-based gene therapies motivate development of more efficient manufacturing processes. Recently, the US Food and Drug Administration (FDA) approved the first rAAV-based gene therapy product manufactured in the baculovirus expression vector system (BEVS), a technology that demonstrated production of high titers of full capsids. This work presents a first mechanistic model describing the key extracellular and intracellular phenomena occurring during baculovirus infection and rAAV maturation in the BEVS. The model predictions are successfully validated for in-house and literature experimental measurements of the vector genome and of structural and non-structural proteins collected during rAAV manufacturing in the BEVS with the TwoBac and ThreeBac constructs. A model-based analysis of the process is carried out to identify the bottlenecks that limit full capsid formation. Vector genome amplification is found to be the limiting step for rAAV production in Sf9 cells using either the TwoBac or ThreeBac system. In turn, vector genome amplification is hindered by limiting Rep78 levels. Transgene and non-essential baculovirus protein expression in the insect cell during rAAV manufacturing also negatively influences the rAAV production yields.
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Assuring that cell therapy products are safe before releasing them for use in patients is critical. Currently, compendial sterility testing for bacteria and fungi can take 7-14 days. The goal of this work was to develop a rapid untargeted approach for the sensitive detection of microbial contaminants at low abundance from low volume samples during the manufacturing process of cell therapies. We developed a long-read sequencing methodology using Oxford Nanopore Technologies MinION platform with 16S and 18S amplicon sequencing to detect USP <71> organisms and other microbial species. Reads are classified metagenomically to predict the microbial species. We used an extreme gradient boosting machine learning algorithm (XGBoost) to first assess if a sample is contaminated, and second, determine whether the predicted contaminant is correctly classified or misclassified. The model was used to make a final decision on the sterility status of the input sample. An optimized experimental and bioinformatics pipeline starting from spiked species through to sequenced reads allowed for the detection of microbial samples at 10 colony-forming units (CFU)/mL using metagenomic classification. Machine learning can be coupled with long-read sequencing to detect and identify sample sterility status and microbial species present in T-cell cultures, including the USP <71> organisms to 10 CFU/mL. IMPORTANCE This research presents a novel method for rapidly and accurately detecting microbial contaminants in cell therapy products, which is essential for ensuring patient safety. Traditional testing methods are time-consuming, taking 7-14 days, while our approach can significantly reduce this time. By combining advanced long-read nanopore sequencing techniques and machine learning, we can effectively identify the presence and types of microbial contaminants at low abundance levels. This breakthrough has the potential to improve the safety and efficiency of cell therapy manufacturing, leading to better patient outcomes and a more streamlined production process.
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Some members of MIT's Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) previously published content on the "Quality Risk Management in the Context of Viral Contamination", which described tools, procedures, and methodologies for assessing and managing the risk of a potential virus contamination in cell culture processes. To address the growing industry interest in moving manufacturing toward open ballrooms with functionally closed systems and to demonstrate how the ideas of risk management can be leveraged to perform a risk assessment, CAACB conducted a case study exercise of these new manufacturing modalities. In the case study exercise, a cross-functional team composed of personnel from many of CAACB's industry membership collaboratively assessed the risks of viral cross-contamination between a human and non-human host cell system in an open manufacturing facility. This open manufacturing facility had no walls to provide architectural separation of two processes occurring simultaneously, specifically a recombinant protein perfusion cell culture process using the human cell line, HEK-293 (Process 1) and a downstream postviral filtration unit operation (Process 2) of a recombinant protein produced in CHO cells. This viral risk assessment focused on cross-contamination of the Process 2 filtration unit operation after the Process 1 perfusion bioreactor was contaminated with a virus that went undetected. The workflow for quality risk management that is recommended by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) was followed, which included identifying and mapping the manufacturing process, defining the risk question, risk evaluation, and risk control. The case study includes a completed Failure Mode and Effects Analysis (FMEA) to provide descriptions of the specific risks and corresponding recommended risk reduction actions.
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Gestão de Riscos , Vírus , Cricetinae , Animais , Humanos , Cricetulus , Células HEK293 , Medição de Risco , Proteínas RecombinantesRESUMO
Because covalent chemistry can diminish the optical and electronic properties of single-walled carbon nanotubes (SWCNTs), there is significant interest in developing methods of controllably functionalizing the nanotube sidewall. To date, most attempts at obtaining such control have focused on reaction stoichiometry or strength of oxidative treatment. Here, we examine the role of surfactants in the chemical modification of single-walled carbon nanotubes with aryl diazonium salts. The adsorbed surfactant layer is shown to affect the diazonium derivatization of carbon nanotubes in several ways, including electrostatic attraction or repulsion, steric exclusion, and direct chemical modification of the diazonium reactant. Electrostatic effects are most pronounced in the cases of anionic sodium dodecyl sulfate and cationic cetyltrimethylammonium bromide, where differences in surfactant charge can significantly affect the ability of the diazonium ion to access the SWCNT surface. For bile salt surfactants, with the exception of sodium cholate, we find that the surfactant wraps tightly enough such that exclusion effects are dominant. Here, sodium taurocholate exhibits almost no reactivity under the explored reaction conditions, while for sodium deoxycholate and sodium taurodeoxycholate, we show that the greatest extent of reaction is observed among a small population of nanotube species, with diameters between 0.88 and 0.92 nm. The anomalous reaction of nanotubes in this diameter range seems to imply that the surfactant is less effective at coating these species, resulting in a reduced surface coverage on the nanotube. Contrary to the other bile salts studied, sodium cholate enables high selectivity toward metallic species and small band gap semiconductors, which is attributed to surfactant-diazonium coupling to form highly reactive diazoesters. Further, it is found that the rigidity of anionic surfactants can significantly influence the ability of the surfactant layer to stabilize the diazonium ion near the nanotube surface. Such Coulombic and surfactant packing effects offer promise toward employing surfactants to controllably functionalize carbon nanotubes.
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Compostos de Diazônio/química , Nanotubos de Carbono , Tensoativos/química , Adsorção , Simulação de Dinâmica Molecular , SaisRESUMO
Excessive sample volumes continue to be a major limitation in the analysis of protein-protein interactions, motivating the search for label-free detection methods of greater sensitivity. Herein, we report the first chemical approach for selective protein recognition using fluorescent single-walled carbon nanotubes (SWNTs) enabling label-free microarrays capable of single protein detection. Hexahistidine-tagged capture proteins directly expressed by cell-free synthesis on SWNT/chitosan microarray are bound to a Ni(2+) chelated by Nα,Nα-bis(carboxymethyl)-L-lysine grafted to chitosan surrounding the SWNT. The Ni(2+) acts as a proximity quencher with the Ni(2+)/SWNT distance altered upon docking of analyte proteins. This ability to discern single protein binding events decreases the apparent detection limit from 100 nM, for the ensemble average, to 10 pM for an observation time of 600 s. This first use of cell-free synthesis to functionalize a nanosensor extends this method to a virtually infinite number of capture proteins. To demonstrate this, the SWNT microarrays are used to analyze a network of 1156 protein-protein interactions in the staurosporine-induced apoptosis of SH-SY5Y cells, confirming literature predictions.
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Fluorescência , Nanotubos de Carbono/química , Análise Serial de Proteínas , Proteínas/análise , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Quitosana/química , Humanos , Nanotecnologia , Níquel/química , Tamanho da Partícula , Ligação Proteica , Estaurosporina/farmacologia , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
Food systems of the future will need to face an increasingly clear reality - that a protein-rich diet is essential for good health, but traditional meat products will not suffice to ensure safety, sustainability, and equity of food supply chains at a global scale. This paper provides an in-depth analysis of bioprocess technologies needed for cell-based meat production and challenges in reaching commercial scale. Specifically, it reviews state-of-the-art bioprocess technologies, current limitations, and opportunities for research across four domains: cell line development, cell culture media, scaffolding, and bioreactors. This also includes exploring innovations to make cultured meat a viable protein alternative across numerous key performance indicators and for specific applications where traditional livestock is not an option (e.g., local production, space exploration). The paper explores tradeoffs between production scale, product quality, production cost, and footprint over different time horizons. Finally, a discussion explores various factors that may impact the ability to successfully scale and market cultured meat products: social acceptance, environmental tradeoffs, regulatory guidance, and public health benefits. While the exact nature of the transition from traditional livestock to alternative protein products is uncertain, it has already started and will likely continue to build momentum in the next decade.
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Abastecimento de Alimentos , Carne , Reatores BiológicosRESUMO
Controlling microbial risks in cell therapy products (CTPs) is important for product safety. Here, we identified the nicotinic acid (NA) to nicotinamide (NAM) ratio as a biomarker that detects a broad spectrum of microbial contaminants in cell cultures. We separately added six different bacterial species into mesenchymal stromal cell and T cell culture and found that NA was uniquely present in these bacteria-contaminated CTPs due to the conversion from NAM by microbial nicotinamidases, which mammals lack. In cells inoculated with 1 × 104 CFUs/mL of different microorganisms, including USP <71> defined organisms, the increase in NA to NAM ratio ranged from 72 to 15,000 times higher than the uncontaminated controls after 24 h. Importantly, only live microorganisms caused increases in this ratio. In cells inoculated with 18 CFUs/mL of Escherichia coli, 20 CFUs/mL of Bacillus subtilis, and 10 CFUs/mL of Candida albicans, significant increase of NA to NAM ratio was detected using LC-MS after 18.5, 12.5, and 24.5 h, respectively. In contrast, compendial sterility test required >24 h to detect the same amount of these three organisms. In conclusion, the NA to NAM ratio is a useful biomarker for detection of early-stage microbial contaminations in CTPs.
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We report the selective detection of single nitric oxide (NO) molecules using a specific DNA sequence of d(AT)(15) oligonucleotides, adsorbed to an array of near-infrared fluorescent semiconducting single-walled carbon nanotubes (AT(15)-SWNT). While SWNT suspended with eight other variant DNA sequences show fluorescence quenching or enhancement from analytes such as dopamine, NADH, L-ascorbic acid, and riboflavin, d(AT)(15) imparts SWNT with a distinct selectivity toward NO. In contrast, the electrostatically neutral polyvinyl alcohol enables no response to nitric oxide, but exhibits fluorescent enhancement to other molecules in the tested library. For AT(15)-SWNT, a stepwise fluorescence decrease is observed when the nanotubes are exposed to NO, reporting the dynamics of single-molecule NO adsorption via SWNT exciton quenching. We describe these quenching traces using a birth-and-death Markov model, and the maximum likelihood estimator of adsorption and desorption rates of NO is derived. Applying the method to simulated traces indicates that the resulting error in the estimated rate constants is less than 5% under our experimental conditions, allowing for calibration using a series of NO concentrations. As expected, the adsorption rate is found to be linearly proportional to NO concentration, and the intrinsic single-site NO adsorption rate constant is 0.001 s(-1) µM NO(-1). The ability to detect nitric oxide quantitatively at the single-molecule level may find applications in new cellular assays for the study of nitric oxide carcinogenesis and chemical signaling, as well as medical diagnostics for inflammation.
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DNA/química , Nanotubos de Carbono , Óxido Nítrico/química , Adsorção , Fluorescência , Microscopia de Força Atômica , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
There has been renewed interest in solar concentrators and optical antennas for improvements in photovoltaic energy harvesting and new optoelectronic devices. In this work, we dielectrophoretically assemble single-walled carbon nanotubes (SWNTs) of homogeneous composition into aligned filaments that can exchange excitation energy, concentrating it to the centre of core-shell structures with radial gradients in the optical bandgap. We find an unusually sharp, reversible decay in photoemission that occurs as such filaments are cycled from ambient temperature to only 357 K, attributed to the strongly temperature-dependent second-order Auger process. Core-shell structures consisting of annular shells of mostly (6,5) SWNTs (E(g)=1.21 eV) and cores with bandgaps smaller than those of the shell (E(g)=1.17 eV (7,5)-0.98 eV (8,7)) demonstrate the concentration concept: broadband absorption in the ultraviolet-near-infrared wavelength regime provides quasi-singular photoemission at the (8,7) SWNTs. This approach demonstrates the potential of specifically designed collections of nanotubes to manipulate and concentrate excitons in unique ways.
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Nanotubos de Carbono/química , Nanotecnologia , TemperaturaRESUMO
Manufacturing of recombinant adeno-associated virus (rAAV) viral vectors remains challenging, with low yields and low full:empty capsid ratios in the harvest. To elucidate the dynamics of recombinant viral production, we develop a mechanistic model for the synthesis of rAAV viral vectors by triple plasmid transfection based on the underlying biological processes derived from wild-type AAV. The model covers major steps starting from exogenous DNA delivery to the reaction cascade that forms viral proteins and DNA, which subsequently result in filled capsids, and the complex functions of the Rep protein as a regulator of the packaging plasmid gene expression and a catalyst for viral DNA packaging. We estimate kinetic parameters using dynamic data from literature and in-house triple transient transfection experiments. Model predictions of productivity changes as a result of the varied input plasmid ratio are benchmarked against transfection data from the literature. Sensitivity analysis suggests that (1) the poorly coordinated timeline of capsid synthesis and viral DNA replication results in a low ratio of full virions in harvest, and (2) repressive function of the Rep protein could be impeding capsid production at a later phase. The analyses from the mathematical model provide testable hypotheses for evaluation and reveal potential process bottlenecks that can be investigated.
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Recombinant adeno-associated viruses (rAAVs) are among the most important vectors for in vivo gene therapies. With the rapid development of gene therapy, current rAAV manufacturing capacity faces a challenge to meet the emerging demand for these therapies in the future. To examine the bottlenecks in rAAV production during cell culture, we focus here on an analysis of cellular pathways of rAAV production, based on an overview of assembly mechanisms first in the wild-type (wt) AAV replication and then in the common methods of rAAV production. The differences analyzed between the wild-type and recombinant systems provide insights into the mechanistic differences that may correlate with viral productivity. Based on these analyses, we identify potential barriers to high productivity of rAAV and discuss future directions for improvement to meet the emerging needs set by the growth of rAAV-based therapy and the needs of patients.
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Dependovirus , Vetores Genéticos , Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , HumanosRESUMO
The optimization of upstream and downstream processes for production of recombinant adeno-associated virus (rAAV) with consistent quality depends on the ability to rapidly characterize critical quality attributes (CQAs). In the context of rAAV production, the virus titer, capsid content, and aggregation are identified as potential CQAs, affecting the potency, purity, and safety of rAAV-mediated gene therapy products. Analytical methods to measure these attributes commonly suffer from long turnaround times or low throughput for process development, although rapid, high-throughput methods are beginning to be developed and commercialized. These methods are not yet well established in academic or industrial practice, and supportive data are scarce. Here, we review both established and upcoming analytical methods for the quantification of rAAV quality attributes. In assessing each method, we highlight the progress toward rapid, at-line characterization of rAAV. Furthermore, we identify that a key challenge for transitioning from traditional to newer methods is the scarcity of academic and industrial experience with the latter. This literature review serves as a guide for the selection of analytical methods targeting quality attributes for rapid, high-throughput process characterization during process development of rAAV-mediated gene therapies.