Modern Cell-based Assays for Bioprocessing
Nexcelom high-throughput platforms can rapidly quantify cell concentration and viability, as well as cell-based assays to streamline every step in the bioprocessing pipeline from engineering host cells to biological protein production.
The three main types of therapy are:
- Biologic protein-based therapy with monoclonal antibodies (mAbs)
- Gene therapy (e.g., Luxturna)
- Cell therapy (e.g., ex vivo CAR-T cell-based therapies like Kymriah and Yescarta)
Biologic production relies on accurate measurements of total cell concentration and viability at every step to ensure cell survival and effective mAb production. Additional cell fitness characterization can be performed using specific fluorescent reagents such as reactive oxygen species (ROS) or caspase 3/7 apoptosis detection.
Nexcelom products utilize state-of-the-art image cytometry methods to measure total and viable cell populations, ensuring consistency and reproducibility in the bioprocessing workflow.
Optimal biologic production depends on:
- Engineering host cells
- Effective cell transfection
- Optimizing cell culture conditions (media, pH, confluence)
- Minimizing variation
- Developing assays to monitor growth and scale-up biologic production
- Monoclonality verification
The first mAb was created in 1975 after the fusion of a mouse B lymphocyte with a murine myeloma cell; these hybridomas were able to continuously secrete large quantities of antibodies [Joubert et al. 2019]. The 1970s also saw the production of the first genetically engineered human insulin by Escherichia coli.
In 1987, Chinese Hamster Ovary (CHO) cells were introduced to produce mAb, which was a major advancement in mAb manufacturing. This system was used to produce tissue plasminogen activator in 1987, followed by erythropoietin two years later. In the 1990s, the first chimeric recombinant and humanized antibodies were established [Joubert et al. 2019].
Currently, more than half of the available commercial mAbs are made in CHO cells. They offer the advantages of being robust, genetically manipulable, and possessing a strong track record with regulatory authorities.
They can be modified to:
- Improve cell growth
- Minimize apoptosis
- Optimize metabolism
- Promote protein product secretion
Mammalian cells provide the additional benefit of similarity with human glycosylation and other post-translational modifications (PTMs) [Dalton and Barton, 2014]. Approximately 70% of biopharmaceuticals are based on mammalian cell culture processes, which can bottleneck their production due to limits on cultivation time, growth capacity, and product yield [Fischer et al., 2015].
Despite these challenges, mAbs have become one of the most widely used therapeutic classes. They currently account for half of the ten best-selling drugs and $50 billion in sales annually [Baeshen et al. 2014]. While antibody-based drugs are an important tool in the armamentarium against hundreds of medical conditions, several critical biomanufacturing issues are obstacles in the industry, including protein expression platform productivity [Joubert et al. 2020].
This resource will discuss obstacles in the in vitro production of therapeutic proteins including:
- Engineering CHO cells with CRISPR
- Transfecting cell lines
- Selecting media
- Monitoring growth and viability
- Single-cell cloning
- Clone selection
Each of these critical steps relies on accurate measurements of cell concentration and viability. Counting viable cells is necessary for downstream assays of productivity (antibody titer, enzyme activity levels, etc.).