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Leveraging Innovative Transposase Technology for a Highly Reliable and Productive CMC Platform

Leveraging Innovative Transposase Technology for a Highly Reliable and Productive CMC Platform

May 30, 2024PAO-06-24-CL-05
BiomanufacturingChemistry, Manufacturing, and ControlsPortable CMCWheeler BioTransposonsATUMStable Bulk CulturesQ22024Transposase

The Evolving Landscape for Early Cell Line Development

The growing importance of cost-efficiency and speed-to-clinic, along with advances in bioprocessing techniques, are driving innovation in biopharma development and refinement of lead therapeutic candidates. A persistent bottleneck in early development comes with cell line development (CLD), especially generating cultures from single-cell clones. Cloning requires extensive time and resources while burdening drug development innovators with significant risks. Innovators can skip the cloning step, however, and use stable bulk cultures (SBCs). This has emerged as an efficacious method for early-phase lead development and refinement, but there is considerable variation in methodology and results in the development of SBCs. While this variation resulted in some skepticism and delayed utilization of SBCs, significant advances in SBC production techniques have since catalyzed their adoption.1 One method in particular, Leap-In Transposase®, a semi-targeted integration (STI) technique developed and commercialized by ATUM, offers a solution to overcome the challenges posed by other SBC generation methods.

While SBCs have recently grown in popularity for accelerating early biotherapeutic development, they have been extensively studied and implemented for nearly a decade. SBCs emerged in scientific literature long before the COVID-19 pandemic, establishing a scientifically sound foundation that enabled unprecedented development timelines during the pandemic response. Multiple large pharmaceutical companies and global CDMOs have produced preclinical toxicology supplies and early clinical trial materials using SBCs based on years of academic research demonstrating process consistency, product quality attributes, and comparability to derivatized clonal cell lines.

Significant clinical safety and efficacy data was accumulated across the COVID-19 pandemic, which correlates well with the previously established scientific groundwork on SBCs. In its February 2021 guidance for developing SARS-CoV-2–neutralizing monoclonal antibodies (mAbs), the FDA explicitly supported using SBCs instead of clonally derived cell lines for early clinical batch production. The successful real-world implementation of SBCs during the pandemic has led to broader consideration of their utility. Over 50% of leading biopharma companies have acknowledged interest in leveraging SBCs to produce toxicology supplies and potentially early clinical materials within the next five years across their non-COVID pipelines.2

The Challenges of Traditional Cell Line Development

The use of clonal cell lines for manufacturing comes from the FDA guidance that "the growth pattern and morphological appearance of the cell line should be determined and should be stable from the master cell bank to the end-of-production cells."3 It was later determined by standard practice that the best way to ensure a culture remained stable was to demonstrate that all of the cells in a production culture are genetically identical (clonal). With the advent of new methods of generating cell lines, it is possible to demonstrate stability of a culture that consists of a pool of genetically diverse cells, as long as the pool is generated so that the cells continue to behave similarly throughout production.

To appreciate the accelerative benefits of SBCs, it is informative to consider the laborious, time-intensive process of traditional clonal CLD. CLD requires substantial infrastructure, expertise, and specialized tools to isolate high-producing recombinant clones from heterogeneous transfected pools through multiple rounds of single-cell isolation, screening, and selection.

Generation of a clonal cell line is achieved by depositing a single cell per well in a 96- or 384-well dish. This step is often accomplished using one of several different types of sorting and imaging equipment. Thousands of individual cells are then cultured over multiple weeks, passaging them into new wells regularly and screening them regularly for productivity. Growth and sterility are managed through automated instrumentation. Growing and passaging the clones is a multi-week process that requires simultaneous characterization and heavy documentation, mostly through the use of thousands of images spread over thousands of individual cultures. The work of managing the growth, maintaining sterility, tracking the progress of the clones, and correlating their images is handled through specialized automated instrumentation. Resulting clones are then extensively characterized to identify elite producers exhibiting desirable phenotypic traits. This requires additional instrumentation for assessing growth kinetics, productivity, product quality attributes, and stability. From thousands of clones screened, a small subset of top candidates is banked as primary working cell banks. Final candidates must also be assessed in long-term cultures to demonstrate the stable growth characteristics required by the FDA guidance. In all, a complete CLD process and stability assessment can extend across months to almost a year, depending on molecule complexity.4

Realizing the Potential of Stable Bulk Cultures

SBCs offer a means to dramatically accelerate early CLD by deferring single-cell cloning efforts to post-IND stages. As pools of recombinant cells, SBCs circumvent clonal isolation and screening, enabling rapid process characterization, product quality evaluations, toxicology study production, and manufacturing of early clinical materials. When appropriately controlled, SBCs deliver suitably homogeneous phenotypes to satisfy regulatory guidance as reliable cell substrates.

Since SBCs still undergo transfection, selection, and amplification steps akin to early phase CLD, their ultimate success hinges on the recombinatorial tools enabling stable genomic integration.

Achieving a Challenging Baseline – Stable Transfection

All mAb therapies are derived from cells that have been successfully transfected such that their modifications are passed from one cell generation to the next without loss of gene of interest (GOI) expression or other degradations to the cells' ability to produce sufficiently high titers of the target protein. The standard method for achieving this has been to generate a stable cell line through plasmid transfection.

This process has plagued both innovators and manufacturers of mAb therapies due to the scientific and technical challenges that must be carefully navigated to ensure the successful expression of therapeutic proteins. The complexity in achieving successful plasmid transfection stems from the inherent nature of genomic integration and the technical limitations of current methodologies.4,5

One of the foremost challenges in achieving successful chromosomal integration of a GOI through plasmid transfection is the low efficiency of random integration events and the difficulty in precisely targeting desired genomic locations. The efficiency of random integration via plasmid delivery of GOIs is typically low because only a small fraction of transfected cells will successfully integrate the foreign DNA into their genome in a manner that allows for stable expression.6 Furthermore, even when integration does occur, the lack of control over the insertion site can lead to variable expression outcomes due to the position effect, where the genomic context influences the activity of the integrated gene.

The position effect is a significant challenge because the GOI's expression level can be heavily influenced by its integration site within the genome. Integration into heterochromatin, for example, can lead to gene silencing, while integration into euchromatin might result in high expression levels. Moreover, the integration process can disrupt host genes or regulatory elements, potentially leading to genomic instability or unintended phenotypic changes in the host cells.7

Considering, then, the inherent issues in gene integration stability and predictability when using plasmid transfection to incorporate a GOI into a pool of cells through random integration, this method is unsuitable for creating reliable, productive SBCs for use in lead selection and materials scaling in a mAb therapy development process.

Methods for SBC Development

Developing SBCs requires the necessary transfection, selection, and amplification steps that are common to CLD, but they are distinguished by the methods used for stable genetic integration compared with the random integration method used in transient plasmid transfection.6 The primary techniques utilized in SBC generation utilize either Targeted or Semi-Targeted Integration.

Targeted Integration Methods

Researchers have employed site-directed integration techniques using specific recombinases like the Cre/loxP system, Flp/FRT system, and phiC31/R4 integrases to overcome the challenges of random gene insertion. These methods aim for controlled insertion of transgenes into predetermined genomic locations. However, they require the prior establishment of platform cell lines that already have a recombination site inserted into specific genomic regions.

The recent elucidation and publication of CHO cell line genomes has opened the door to precise genome engineering using engineered nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas system. These technologies allow for targeted mutations or specific sequence modifications, relying on the cell's DNA repair mechanisms, notably nonhomologous end-joining (NHEJ) and homology-directed repair (HDR). While NHEJ is more error-prone and often used for gene knockouts, HDR can achieve precise modifications with the help of homologous DNA sequences. Historically, CHO cells have been less amenable to homology-based integration, limiting the use of HDR in favor of NHEJ-based methods.

The CRISPR/Cas9 system, derived from a bacterial immune defense mechanism, simplifies genome editing. It uses engineered RNA molecules to guide the Cas9 enzyme to specific DNA locations, allowing for precise edits. This system is advantageous for its simplicity, efficiency, and low cost compared to earlier protein-based editing tools. Recent advancements have improved the CRISPR/Cas9 system's effectiveness in CHO cells, demonstrating high efficiency in inducing targeted gene edits.9

While the above methods of targeted integration are undeniably powerful in their precision, their use for generating stable bulk cultures is hampered by significant challenges:

  • Limited editable sites: Despite advances in genome sequencing, our understanding of the CHO cell genome and its regulation is incomplete. There may be limited 'safe harbors' or genomic sites where transgenes can be inserted without disrupting essential genes or regulatory elements10 Finding and validating these sites for every new mAb therapy significantly limits the applicability of targeted integration.

  • Intellectual property and accessibility: Certain genome editing technologies are often protected by patents and intellectual property rights, which can restrict access to these technologies or increase the cost of developing new therapies, especially for smaller biotech companies or academic institutions.11

  • Unpredictability of complex biological systems: The expression of mAbs (or any other recombinant protein) is influenced by many factors beyond the site of gene integration, including chromatin structure, regulatory element interactions, and metabolic burden on the host cell. Even with targeted integration, achieving consistently high yields of adequately folded and glycosylated antibodies can be unpredictable.12

  • Technological evolution: The rapid pace of biotechnological innovation means that today's cutting-edge techniques may be superseded by even more efficient or straightforward methods tomorrow. Investing heavily in a particular technology might risk obsolescence, making it a less attractive option for long-term therapeutic development.

Semi-Targeted Integration Methods

To enhance the efficiency, reliability, and stability of cell lines used for producing biopharmaceuticals, scientists have shifted toward a more controlled method of inserting genes, known as semi-targeted integration, facilitated by transposase systems. This approach is rapidly becoming preferred over the traditional random integration for creating cell lines and is particularly advantageous for producing SBCs. Transposase technologies utilize a dual-component system, which includes an active transposase enzyme and a transposon vector. This vector carries not only the gene for the therapeutic protein but also a selectable marker, flanked by specific DNA sequences that the transposase recognizes to insert the gene into the host cell's genome through a precise "cut-and-paste" mechanism. Such technologies often target gene insertion into areas of the genome that are active and accessible, leading to the insertion of single, intact copies of the gene, resulting in cell lines that are more genetically stable.

Furthermore, transposase technology has shown promise in improving the selection of cell lines that produce therapeutic proteins, enabling the maintenance of quality comparable to their original cell pools. Sustaining consistency with the original cell pools is particularly beneficial in developing biotherapeutic drugs, offering consistency in drug substances produced from both stable cell pools and individual clones. Such consistency is crucial for the seamless transition from early clinical studies to later clinical trials and commercial production stages.

Various transposase systems, each with unique properties and efficiencies, have been adapted for use in mammalian cells, including the Leap-In Transposase® and piggyBac® systems. These tools have been engineered or selected for higher performance and specific integration preferences. The introduction of transposase technology as a modular addition to existing cell line development platforms allows for rapid adoption without significant changes to the workflow, offering a faster path to clinical trials for new therapies.13

Leap-In Transposase®: The Power of Transposon–Transposase Pairs

ATUM's Leap-In Transposase® system is a groundbreaking approach that significantly streamlines the development of stable cell lines. This system, when combined with the capabilities of the VectorGPS® platform, represents a comprehensive solution for the rapid and efficient production of biologics, addressing critical challenges in the biotech industry. ATUM's Leap-In Transposase® technology leverages a synthetic transposon and transposases designed by machine learning at ATUM. The optimized system ensures the integration of intact DNA constructs into active chromatin regions, minimizing the risk of construct rearrangement and maintaining the stability and expression of the inserted gene. The synthetic transposon contains the expression construct and selection marker flanked by inverted terminal repeats (ITRs). The Leap-In Transposase® recognizes these ITRs, precisely excises the entire transposon, and integrates it with high fidelity into transcriptionally active sites across the genome, enhancing the potential for high-level gene expression.

Leap-In Transposase® was launched in 2018 and has since been used in over 150 projects via ATUM's service business and deployed to over 40 active licensees, ranging from large pharma to emerging biotech companies. With 22 regulatory filings across three jurisdictions in the past three years, including for phase II/III trials, this is the leading, industry-standard transposase platform. Likewise, the platform is very well suited for large, complex, and routine molecules, including traditional proteins, mAbs, and fusion proteins, as well as the various multi-specific antibody formats, and it can be applied within a wide range of host cell types.14

VectorGPS®: Custom Design and Optimization

The VectorGPS® platform is ATUM's answer to the nuanced challenges of protein expression in biotechnology. It facilitates the parallel design and synthesis of multiple synthetic transposons, which allows for the fine-tuning of expression levels of protein subunits, ensuring optimal ratios for effective protein assembly and function.

By integrating the Leap-In Transposase® system with the VectorGPS® platform, biotech innovators can reduce process development timelines by enabling the rapid generation of stable pools and the early initiation of process and analytical development. This efficiency is due to the technology's ability to produce similar cells with highly comparable productivity and product quality, streamlining the progression from transfection to material preparation for toxicology studies. Furthermore, the system's capability to maintain genetic and functional stability, alongside high expression levels, ensures the consistent production of high-quality biologics.15

Leveraging Leap-In Transposase® with Portable CMC®

Minimizing the gap between discovery, cell line development, and process development can significantly increase the likelihood of a drug candidate advancing into first-in-human studies and succeeding in later-stage clinical trials. Wheeler Bio believes a different CDMO model is needed for innovators to bring new therapeutic leads to the clinic faster, without the risk and cost associated with traditional outsourcing models. Their flagship service offering, Portable CMC®, is a suite-ready drug substance manufacturing platform that customers can transfer to any CDMO worldwide without royalties, licensing fees, or penalties. By overlapping with the discovery workflow, Portable CMC® reduces timelines and regulatory risks by introducing quality-by-design (QbD) principles earlier in the drug development life cycle, increasing the opportunity for clinical success.16

Wheeler leverages the Leap-In Transposase®-mediated gene delivery system to enable a cost-effective, easily implemented cell line CHO pool generation workflow, delivering highly productive, non-clonal cell lines with consistent pool-to-clone product quality. The resulting data aid in selecting top candidates for further CMC development and clinical manufacturing while also providing preclinical materials in milligram quantities (50 mg of protein A–purified material for four or more mAb candidates). Wheeler's high-productivity cell lines and transposon-based gene delivery reduce quality risks and facilitate a smooth transition from lead selection to clinical supply manufacturing, ultimately accelerating CLD activities by months.

Biotech innovators stand to benefit tremendously from cell line innovation that provides enhanced efficiency without sacrificing quality. As a transformative genome engineering solution for controlled, rapid SBC generation, Leap-In Transposase® has emerged as a vital accelerator. By integrating Leap-In capabilities into its automated and openly accessible Portable CMC® platform, Wheeler Bio empowers innovators with an accelerated path into first-in-human trials by establishing portable, drug-substance CMC support for unique lead candidates. This alliance helps shape the biotherapeutic development future by removing persistent obstacles that drain valuable early-phase time and resources.16

References

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