Introduction
The biopharmaceutical industry witnessed significant innovation, with many new recombinant protein therapeutics receiving FDA approval. However, these therapies are often costly, emphasizing the need for greater efficiency and productivity. Optimizing cell line development (CLD) and upstream processing can boost product quantity and titer, while improvement in downstream steps can enhance product quality.
SynWeave™ is a platform developed by the Biopharmaceutical Development team to enhance manufacturability and efficiency of protein production. It combines state-of-the-art cell line development using transposon-based technologies seamlessly integrated with advanced process development capabilities.
Challenges in cell line development
CHO (Chinese Hamster Ovary) cells are used in therapeutic protein production as they grow in suspension cultures, adapt to various growth conditions, and perform post-translational modifications essential for functional therapeutic proteins for 50 years in industry. However, traditional CLD in CHO generally faces two major challenges: heterogeneous gene expression and low integration efficiency caused by positional effects.
Approaches to mitigate the position effect
A transposon1 is a mobile DNA element that can integrate the entire construct at multiple locations in the genome through a cut-and-paste mechanism. Therefore, transposon-mediated, semi-targeted integration mitigated the challenge of the position effect that caused heterogeneous gene expression and improved the integration efficiency, yielding consistent gene expression. This approach also reduced the labor-intensive step of clones needing screening to identify the best producer/s, as most cells receive a fully functional construct. The timeline for developing stable cell lines is reduced, and high-yielding clones with the desired qualities are generated.
Coupling the clone selection process at an early stage to a robust upstream platform resulted in picking the clones, which helped in enhancing the titer.
Effectiveness of the upstream platforms
The upstream platform integration helps to optimize and improve titers with minimum experiments. The upstream development processes will enable us to generate high titer with stringent quality attributes.
The Next Generation Mammalian Cell Line Development (Figure 1) process was designed to achieve stable clones in 27 weeks. The following steps are conducted.
Monoclone
Monoclonality analysis was carried out as desired by regulatory agencies.
Purification and Product Quality
analysis Top selected monoclones (based on titer) are also evaluated for key quality attributes like size, charge, glycan profile, and activity; thereafter, generation stability was performed for selected ones.
Clone screening
As a part of process development, we use a high throughput Ambr 250 system miniaturized bioreactor to screen the best producers in a real manufacturing-controlled environment. The integration of Design of Experiments (DoE) for parametric and media/feed screening and Process Analytical Technology (PAT) within the Quality by Design (QbD) framework further enhanced the effectiveness of upstream platforms. This approach enabled real-time monitoring and analysis of critical quality attributes, allowing biopharmaceutical companies to maintain steady nutrient levels in bioreactors and optimize process control and efficiency. Robust manufacturing process design space was established using the AMBR 250 system and statistical tools. Design space was tested at a 10L scale before being scaled to larger manufacturing batches.
Ensuring safety, quality and regulatory compliance is key to biologics production and a well-understood cell line plus upstream platform reduced any risk of not meeting the compliance expectations
Key advantages:
- It saved up to 10 weeks compared to traditional methods, streamlining the process
- Increased productivity: Achieved high titer levels of up to 10 g/L with optimized processes
- Versatile application: Supported many products, including mAbs, biosimilars, bispecifics, fusion protein, and other recombinant proteins
Success story
- Both the enhancements (Cell line development with upstream platform processes), when integrated with SynWeave platform, produced a tier of 5-7 g/L for both IgG1 and IgG4 molecules (Figure 2)
- A client obtained a titer of 0.8 g/L in 22 days, whereas with Syngene’s platforms a titer of 4.8 g/L was obtained in 17 days
- Biopharma companies can reduce timelines by integrating upstream platforms and adopting disruptive concepts. Syngene is also working on N-1 perfusion with high cell density fermentation to increase the titer further
