As molecule complexity rises, advanced cell line development platforms are giving biopharma companies new tools to overcome expression challenges, improve stability and reach the clinic faster.
Cell line development (CLD) is a field in constant growth. Biopharmaceutical researchers, small and mid-sized biotechs, and large pharmaceutical organizations alike are always searching for the next innovation that can boost cell line productivity, stability, and scalability. That search becomes far more urgent — and far more difficult — when the molecule in question is a bispecific, trispecific, or another atypical format. These complex architectures introduce a unique set of expression and stability hurdles that traditional CLD approaches often struggle to solve.
A flexible development platform, paired with an innovative mindset and an experienced CDMO partner, can mitigate or even eliminate many of the drawbacks typically associated with complex molecule development. Without this combination of technology and experience, cost overruns and timeline delays aren’t just likely — they’re nearly inevitable.
Complex molecules’ path to market can be a maze
Although there is significant overlap in the development of bispecifics, trispecifics, and atypicals, each molecule, program, and the needs of each sponsor remain unique. Different molecular architectures introduce distinct challenges in expression and stability when, ultimately, the goal is to achieve stable expression for production of the therapeutic molecule. Depending on molecule complexity, altering certain translational elements or signal peptides can significantly improve the expression and overall production.
Achieving the correct ratios of specific DNA chains necessary to correctly configure each molecule and ensuring proper functionality through post-translational modifications are key challenges when developing various complex molecules. Past methodologies and platforms have depended primarily on random integration, using multiple integration events to insert the needed DNA fragments.
Special enzymes like transposases can overcome this difficulty by inserting full gene-of-interest DNA sequences (i.e., cassettes) as one to ensure the correct ratios are achieving high-level expression. Further, although atypical molecules can be difficult to produce at a higher titer, transposase can significantly improve production with demonstrated increases up to 10-fold.
Speed and efficiency during CLD are vital, as sponsors often produce numerous iterations of a molecule during antibody discovery. Although AI tools and in silico tools can be applied to narrow those choices to a few viable candidates, each candidate still must be assessed to explore variables like vector design, enhancer elements, and cellular regulatory mechanisms that can optimize that specific molecule. Newer CLD technologies and approaches offer a more effective route to stable pools, whether a sponsor requires higher titers for a single molecule or needs to identify the top candidate among several bispecific variants.
How a platform can turn CLD uncertainty into surety
Chinese hamster ovary (CHO) cells used for creation of many antibody production cell lines are considered to be genetically plastic, meaning that with extended generations of growth the cells have an inherent risk for genetic and phenotypic change, creating the potential for unstable expression of the genes of interest. For example, gene insertion by random integration can incorporate genes anywhere in the CHO genome, including unfavorable configurations such as truncated or concatemerized forms that lead to gene rearrangements or silencing. This is why stability study execution at 30, 60, and/or 90 generations became an industry standard.
Alternatively, semi-targeted integration utilizes a cut-and-paste gene insertion approach that greatly reduces integration variability, making instability less of a concern. While stability will still be confirmed, the reduced risk allows more resources to be allocated to other areas of the program, which helps achieve time and cost savings. An innovative approach and understanding of how CLD technologies can be utilized to best support a project’s success is critical and even more important when choosing to outsource development.
At AGC Biologics, we have implemented a semi-targeted integration via ATUM’s Leap-In Transposase® expression platform. Transposase allows for a high level of integration, preferring areas of open, transcriptionally active chromatin, translating to the likelihood of cell lines that are both stable and highly expressing. The combination of multiple gene of interest integration events across multiple accessible genomic regions leads to a higher probability of more protein/antibody produced per cell. Further, transposase technology fosters the probability of reduced diversity in product quality from pool to clone, enabling early development at the pool level for streamlined transition to clone and final process in many cases.
While each molecule is different, the transposase capabilities allow AGC experts to quickly assess metrics like product quality, scalability, and purification methods or other elements of process development. AGC Biologics has high-throughput screening technology capable of efficiently analyzing more than 6,400 clones at once, versus the sub-1,000-clone capacity of non-automated screening technologies. As screening takes place, customers are consistently updated on their molecule’s performance (or each variant’s performance) in scaling, quality, and titer. In addition to accelerating the customer’s timeline, efficient and transparent screening equips customers with comprehensive data to make well-informed decisions about how best to proceed with a molecule.
Technology advancements will further accelerate and simplify CLD
As molecule complexity increases and the number of viable candidates expands, expression platforms have evolved to keep pace. AGC Biologics and the industry as a whole have achieved shorter timelines through dedication to continuous innovation and improvement. The emergence of high-throughput screening methods and increased process automation have led to more effective titer assays, better product quality assays, and faster turnaround.
Process controls and process analytical technologies have evolved alongside development tools in forms such as real-time monitoring, continuous manufacturing, and, to some extent, advanced perfusion methods. To mitigate contamination risk and minimize associated waste, research focuses on refining single-use technologies, including bioreactors and purification membranes. In terms of molecular methods being used in CLD, copy number analysis is becoming a more popular in-house analytical tool for CDMOs as the necessary instrumentation becomes more accessible and cost-effective to implement. The benefits of bringing next-generation sequencing capabilities in-house include faster turnaround times and earlier access to more expansive datasets.
Finally, and not unexpectedly, an increasing use of AI can be expected in data management, data compiling, and data analysis. It is already heavily applied to in silico characterization for early prediction of molecule behaviors or properties, and research is being conducted for additional applications such as multi-omics data analysis.
Get in on the ground floor
Developing complex molecules requires tailoring the expression platform and program to overcome challenges unique to each. The sooner a sponsor engages with a CDMO after identifying a promising molecule or variants, the better, as it allows proactive adaptation of platforms and resources. Initial talks should cover the drug type and progress. Customers wanting a specific assay built should verify the CDMO’s experience with their molecule type. Sponsors need to define their strategic goals before engaging, including end goals and a “Plan B” if the molecule underperforms. This strategy helps the CDMO streamline the path to the sponsor’s objectives.
CLD is never a one-size-fits-all approach, especially when working with complex molecules. The CDMO’s facility and platform must be flexible enough to conform to the demands of the molecule. AGC Biologics has worked with a variety of molecule types intended for different markets around the globe. In addition to offering cutting-edge CLD technologies, we offer a diverse network of scientists and project managers who actively engage in reviewing customer proposals and collaboratively resolving the unique challenges associated with each molecule.
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