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    Home > Biochemistry News > Biotechnology News > The new "battle" of cell and gene therapy: how to break through the technological bottleneck?

    The new "battle" of cell and gene therapy: how to break through the technological bottleneck?

    • Last Update: 2021-10-11
    • Source: Internet
    • Author: User
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    Recently, CRISPR pioneer Professor Zhang Feng and his team released new results in "Science", which aroused the industry's new attention to gene therapy
    .
    Finding inspiration from the human body, developing a new mRNA delivery platform to solve the delivery problem, gene therapy has made a new breakthrough in technology

    .

    With the approval of the domestic first CAR-T therapy Fosun Kate's Yikaida® (Akirensai injection), the domestic CAR-T cell therapy has entered the commercialization stage
    .

    The next 3-5 years are expected to usher in an explosive growth phase of the cell and gene therapy drug pipeline.
    However, due to the current complex development process and slow evolution, production and technology have become new problems for pharmaceutical companies to solve

    .

    01 The bottleneck of gene therapy technology: plasmid and virus vector

    01 The bottleneck of gene therapy technology: plasmid and virus vector

    "Plasmids and vectors are the two most critical points in the gene therapy process
    .
    " Yang Hongyan, R&D director of Cytiva Greater China, said in an interview with E medicine manager

    .

    Plasmids are widely present in the biological world and have the ability to replicate independently, so that they can maintain a constant copy number in the progeny cells and express the genetic information they carry
    .
    The most commonly used viral vectors in gene therapy are often produced by packaging after multiple plasmids are transiently transformed.
    Therefore, the process development and industrialization solutions of plasmids and viral vectors are bottlenecks that urgently need to be resolved in the field of gene therapy

    .

    In gene therapy technology, plasmids are often derived from E.
    coli fermentation system

    .
    It takes about 3 to 4 days for E.
    coli strains to recover from entering the reactor for high-density fermentation and then to the lower tank

    .
    "How to achieve stable high-density fermentation as much as possible in a limited time, and at the same time, the expression level is relatively high.
    This is a challenge and the key to the process competition

    .
    Because the higher the expression level of E.
    coli, the higher the batch capacity.
    High, leading to a reduction in the cost of plasmid production,” Yang Hongyan explained

    .

    Escherichia coli grows and replicates very quickly, with a doubling time of about 20 minutes
    .
    In this process, the optimization of the culture medium and feeding strategy, the parameters of the fermentation tank, such as temperature, aeration rate, and stirring speed, will all affect the growth of E.
    coli

    .
    "If these key points of the process are well developed and optimized, and the specific growth rate of E.
    coli is controlled within a reasonable range, the entire fermentation process will be very stable, and the foaming phenomenon in the tank will be relatively controllable

    .
    At present, Cytiva has been established.
    The high-density E.
    coli fermentation process based on disposable reactors has been widely used for large-scale production of plasmids," said Yang Hongyan

    .

    After the fermentation, the collected bacterial cells need to be subjected to alkaline lysis and downstream purification in order to obtain high-quality supercoiled plasmids for future packaging of viral vectors
    .
    The lysis of Escherichia coli is also very challenging.
    How to improve the efficiency and yield of dynamic alkaline lysis in a closed system is the optimization focus of bacterial lysis

    .

    After solving the process challenge of alkaline lysis, another problem ushered in was the establishment of downstream purification processes
    .
    How to remove impurities and obtain high-quality supercoiled plasmids as much as possible is also a problem to be solved in the process

    .
    Under normal circumstances, chromatographic methods are used to remove impurities

    .
    Different plasmids will use different chromatography methods.
    At present, two-step chromatography or three-step chromatography are more commonly used in the industry

    .

    After obtaining high-quality plasmids, what needs to be considered is how to establish a production route for viral vectors
    .
    More than 70% of gene therapy will use viral vectors for the delivery of target genes

    .
    Viral vectors commonly used viruses: lentivirus, adenovirus, adeno-associated virus (the AAV), retrovirus and the like

    .
    Other non-viral vector delivery includes polymers, liposomes, nanomaterials, and emerging exosomes

    .

    Taking the adeno-associated virus vector as an example, its preparation needs to start with cell culture
    .
    About 70% to 80% of viral vectors use HEK293 cell system, and 10% to 20% use insect baculovirus system

    .
    There are currently two ways to cultivate the HEK293 cell system: adherent culture or suspension culture

    .

    In recent years, cell culture technology has gradually shifted from adherent culture to suspension culture in order to expand production capacity in preparation for commercialization
    .
    At the same time, suspension culture can achieve serum-free culture, which is more in line with regulatory requirements

    .

    After the cultured cells reach a certain density, use specific transfection reagents to transfect multiple plasmids into HEK293 cells to package and produce viral vectors
    .
    In the transfection step, the ratio of multiple plasmids, transfection reagents, transfection temperature and time, cell density and other factors will affect the transfection efficiency

    .

    The downstream process of viral vector production includes cell lysis, purification, ultrafiltration concentration, sterilization filtration and canning
    .
    How to improve the recovery rate of viral vectors and the quality of final products in the downstream process routes, such as the removal of empty-shell viruses, also tests future gene therapy companies

    .

    "In response to industry needs, Cytiva has established a research and development and production platform for viral vectors with different serotypes, from cell culture to transfection and downstream purification, to help gene therapy companies promote process development and scale-up production as soon as possible," said Yang Hongyan To
    .

    There are many process links in gene therapy, and each link affects the therapeutic effect, scale and cost after commercialization
    .
    How to produce plasmids and viral vectors more efficiently is one of the keys to controlling the cost of gene therapy

    .

    02 Bottleneck of cell therapy process: a tight window period

    02 Bottleneck of cell therapy process: a tight window period

    As a kind of "living" medicine, cell therapy has high requirements on the process from its production to use
    .

    "China's CAR-T cell therapy has ushered in commercialization.
    The challenge for autologous CAR-T cell therapy lies in the tight window period

    .
    The time from the collection of T cells in the patient to the infusion of CAR-T cells into the patient's body Very limited

    .
    In this limited time to complete CAR-T production, quality inspection release and transportation, there is no room for any mistakes, otherwise the treatment window may be missed for the patient, so the requirements for the CAT-T production process are very high.
    " Yang Hongyan said

    .

    Before the commercialization of CAR-T cell therapy, the preparation of CAR-T cells often used manual processes and open operations, which meant that the dependence on the proficiency of the operator was very high, and at the same time increased the risk of contamination
    .
    CAR-T cells prepared by different operators or even the same operator may have large differences, which makes it difficult to ensure the stability of the process

    .
    As CAR-T cell products enter the commercialization stage, automated process flow and closed operation must be considered to solve the problem of process stability

    .
    The automatic and closed process and the traceability of the whole process data have become the embodiment of the technical strength of each biotechnology company

    .

    Yang Hongyan said, “For Cytiva, the cell drug production process solutions we provide are based on automation and closed operations.
    At the same time, the ChronicleTM digital platform monitors production equipment operation and supply chain logistics information to achieve complete and traceable data throughout the process

    .
    We have also seen that the process development and production of CAR-T cell therapies that are currently on the market use this idea

    .
    "

    03 Enabling and upgrading: talent training, plant construction and supply chain expansion

    Experienced talents are indispensable for process development and optimization.
    However, due to the rapid development of the cell and gene therapy industry, talents are in short supply

    .
    For biotechnology companies, having experienced personnel is the most core competitiveness

    .

    Yang Hongyan introduced: “In the process of cooperating with customers to develop process flow, we will invite customers to implement the process together with us from the first day of the project
    .
    Based on this completely transparent cooperation method, we will Share development thinking and experience with customers and provide training to achieve the goal that they can produce on their own in the future

    .
    For start-up companies, not only can they successfully complete the development of products under research, but also complete the training of talents, and achieve seamless technology connection.
    And transfer

    .
    "

    With China's accession to ICH, the standards for clinical trials and production are gradually in line with international standards, and the rapid construction of GMP factories is also one of the guarantees for commercialization
    .
    However, the construction of a GMP plant is not a simple matter, and it tests the builder's ability to control production standards, future production capacity, and coordination between production lines

    .

    "We will not only train technical personnel, but also provide customers with factory building experience to ensure that they can build a factory that meets the standards in a shorter time and solve product supply problems
    .
    " Yang Hongyan added

    .

    The outbreak of the new crown epidemic in early 2020 and more and more drugs entering commercial production have intensified the demand for equipment and consumables for biopharmaceutical companies.
    At present, the global supply chain is in a very tight state

    .
    It is reported that in order to solve the supply chain problem, Cytiva will continue to invest US$1.
    5 billion globally to expand the production capacity of different products, including culture media, chromatography media, disposable consumables and manpower expansion

    .

    In addition to investing US$1.
    5 billion in the supply chain, Cytiva and E-pharmaceutical managers hosted the "Think Big 2.
    0 Double Choice Platform" Biomedicine Innovation Project Roadshow to help the development of innovative companies in the biomedical industry

    .
    The first sub-station roadshow of the event will be held in Hangzhou Medical Port on September 11, 2021, and the list of Think Big 2.
    0 mentor groups has been announced

    .

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