Comparison of different technologies for producing recombinant adeno-associated virus on a laboratory scale

Adeno-associated virus vectors are among the most promising ones for the delivery of transgenes to various organs and tissues. Recombinant adeno-associated virus (rAAV) is able to transduce both dividing and non-dividing cells, has low immunogenicity, and is able to provide long-term expression of transgenes. Modern technologies make it possible to obtain rAAV for in vivo use, but they are not without drawbacks associated with laboriousness, scalability difficulties, and high cost, therefore, improvement of technological schemes for obtaining rAAV is an urgent issue. The aim of the study was to compare different technological approaches to rAAV production based on different conditions of the transfected HEK293 cell line cultivation on a laboratory scale. Materials and methods: HEK293 cell culture, AAV-DJ Packaging System, PlasmidSelect Xtra Starter Kit were used in the study. The technologies were compared using a model rAAV vector with a single-domain antibody transgene fused to the Fc-fragment of IgG1 specific to botulinum toxin. HEK293 cells were transfected with supercoiled plasmid DNA isolated by three-step chromatographic purification. The identity of the rAAV preparation was determined by electrophoresis, immunoblotting, and real-time polymerase chain reaction. Results: the study demonstrated the efficiency of the chromatographic method for obtaining a supercoiled form of plasmid DNA that can be used for efficient transfection of cell culture in order to produce rAAV. The study compared the following processes of rAAV production: using transient transfection and cultivation of the transfected HEK293 cell suspension in Erlenmeyer flasks, adherent culture in T-flasks, and adherent culture in a BioBLU 5p bioreactor on a matrix of Fibra-Cel disks. Conclusions: the data obtained showed the possibility of using the described approaches to purification of plasmid DNA, cell transfection, and cultivation of the transfected cells under various conditions to obtain rAAV samples that expresses the antibody gene. The BioBLU 5p reactor with Fibra-Cel discs was used for the first time to produce preparative quantities of rAAV on a laboratory scale, which increased the adherent surface area during cell culture and transfection, and, as a result, increased the yield of the target product

Сравнение различных технологий получения рекомбинантного аденоассоциированного вируса в лабораторном масштабе

Adeno-associated virus vectors are among the most promising ones for the delivery of transgenes to various organs and tissues. Recombinant adeno-associated virus (rAAV) is able to transduce both dividing and non-dividing cells, has low immunogenicity, and is able to provide long-term expression of transgenes. Modern technologies make it possible to obtain rAAV for in vivo use, but they are not without drawbacks associated with laboriousness, scalability difficulties, and high cost, therefore, improvement of technological schemes for obtaining rAAV is an urgent issue. The aim of the study was to compare different technological approaches to rAAV production based on different conditions of the transfected HEK293 cell line cultivation on a laboratory scale. Materials and methods: HEK293 cell culture, AAV-DJ Packaging System, PlasmidSelect Xtra Starter Kit were used in the study. The technologies were compared using a model rAAV vector with a single-domain antibody transgene fused to the Fc-fragment of IgG1 specific to botulinum toxin. HEK293 cells were transfected with supercoiled plasmid DNA isolated by three-step chromatographic purification. The identity of the rAAV preparation was determined by electrophoresis, immunoblotting, and real-time polymerase chain reaction. Results: the study demonstrated the efficiency of the chromatographic method for obtaining a supercoiled form of plasmid DNA that can be used for efficient transfection of cell culture in order to produce rAAV. The study compared the following processes of rAAV production: using transient transfection and cultivation of the transfected HEK293 cell suspension in Erlenmeyer flasks, adherent culture in T-flasks, and adherent culture in a BioBLU 5p bioreactor on a matrix of Fibra-Cel disks. Conclusions: the data obtained showed the possibility of using the described approaches to purification of plasmid DNA, cell transfection, and cultivation of the transfected cells under various conditions to obtain rAAV samples that expresses the antibody gene. The BioBLU 5p reactor with Fibra-Cel discs was used for the first time to produce preparative quantities of rAAV on a laboratory scale, which increased the adherent surface area during cell culture and transfection, and, as a result, increased the yield of the target product.Векторы на основе аденоассоциированного вируса являются одними из наиболее перспективных для доставки трансгенов в различные органы и ткани. Рекомбинантный аденоассоциированный вирус (rAAV) способен трансдуцировать как делящиеся, так и неделящиеся клетки, обладает низкой иммуногенностью и способен обеспечивать долгосрочную экспрессию трансгенов. На сегодняшний день существуют технологии, позволяющие получать rAAV для применения in vivo, однако они не лишены недостатков, связанных с трудоемкостью, сложностями масштабирования и высокой стоимостью, поэтому вопрос об усовершенствовании технологических схем получения rAAV является актуальным. Цель работы: сравнение технологических подходов к получению rAAV, основанных на различных условиях культивирования трансфицированной клеточной линии HEK293 в лабораторном масштабе. Материалы и методы: в исследовании использовали культуру клеток HEK293, плазмидную систему AAV-DJ Packaging System, систему PlasmidSelect Xtra Starter Kit. В качестве модели для сравнения технологий использовали вектор rAAV с трансгеном однодоменного антитела, слитого с Fc-фрагментом IgG1, специфичного к ботулотоксину. Применяли метод трансфекции клеток HEK293 суперскрученной плазмидной ДНК, выделенной при помощи трехступенчатой хроматографической очистки. Определение подлинности препарата rAAV проводили методами электрофореза, иммуноблоттинга и полимеразной цепной реакции в режиме реального времени. Результаты: продемонстрирована эффективность получения суперскрученной формы плазмидной ДНК, применимой для эффективной трансфекции с целью получения rAAV. Проведено сравнение процесса транзиентной трансфекции и культивирования трансфицированных клеток HEK293 в условиях суспензии в колбах, адгезии в культуральных флаконах и адгезии в биореакторе BioBLU 5p на матрице из дисков Fibra-Cel с целью продукции rAAV. Выводы: показана возможность применения описанных подходов к очистке плазмидной ДНК, трансфекции и культивированию трансфицированных клеток в различных условиях для получения препарата rAAV, эспрессирующего ген антитела. Реактор BioBLU 5p с дисками Fibra-Cel был впервые использован для получения препаративных количеств rAAV в лабораторном масштабе, что позволило увеличить площадь поверхности адгезии при культивировании и трансфекции клеток и, как следствие, увеличить выход целевого продукта

rAAV expressing recombinant antibody for emergency prevention and long-term prophylaxis of COVID-19

IntroductionNumerous agents for prophylaxis of SARS-CoV-2-induced diseases are currently registered for the clinical use. Formation of the immunity happens within several weeks following vaccine administration which is their key disadvantage. In contrast, drugs based on monoclonal antibodies, enable rapid passive immunization and therefore can be used for emergency pre- and post-exposure prophylaxis of COVID-19. However rapid elimination of antibody-based drugs from the circulation limits their usage for prolonged pre-exposure prophylaxis.MethodsIn current work we developed a recombinant adeno-associated viral vector (rAAV), expressing a SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibody P2C5 fused with a human IgG1 Fc fragment (P2C5-Fc) using methods of molecular biotechnology and bioprocessing.Results and discussionsA P2C5-Fc antibody expressed by a proposed rAAV (rAAV-P2C5-Fc) was shown to circulate within more than 300 days in blood of transduced mice and protect animals from lethal SARS-CoV-2 virus (B.1.1.1 and Omicron BA.5 variants) lethal dose of 105 TCID50. In addition, rAAV-P2C5-Fc demonstrated 100% protective activity as emergency prevention and long-term prophylaxis, respectively. It was also demonstrated that high titers of neutralizing antibodies to the SARS-CoV-2 virus were detected in the blood serum of animals that received rAAV-P2C5-Fc for more than 10 months from the moment of administration.Our data therefore indicate applicability of an rAAV for passive immunization and induction of a rapid long-term protection against various SARS-CoV-2 variants

THE DEVELOPMENT OF A PRODUCTION SCALE TECHNOLOGY OF RECOMBINANT FOLLICLE STIMULATING HORMONE BIOPROCESS IN CHO CELL LINE

The article discusses the development of a production technique for recombinant follicle-stimulating hormone used in the treatment of infertility, in the culture of Chinese hamster ovary cells. As part of the creation of the technological platform receiving hormone the bioprocess in wave-induced motion bioreactor has been optimized. A number of parameters that influence on the bioprocess was studied, as follows: optimal cell density, concentration of metabolites, temperature shift and its duration

PHARMACEUTICAL DEVELOPMENT OF FORMULATION BASED ON BIOPHARMACEUTICAL PREPARATIONS FOR GENE THERAPY

Biopharmaceuticals intended for gene therapy are mostly presented by supercoiled plasmid DNA obtained with bacteria cell lines. The properties of their formulations, including stability under different storage conditions, are barely studied. This article describes the typical pharmaceutical development of freeze-dried gene therapeutics. By means of process analytical technological methods the preparations formulated were controlled during storage. Furthermore, the composition of the formulations was optimized for prolonged storage