Recent advances in breast cancer cell line research.

Breast cancer, a formidable global health challenge, needs continuous translational research to understand the complexity of mechanisms and improve therapeutic and diagnostic strategies. Breast cancer cell lines are of paramount importance as they significantly contribute to the initial stage of research to understand cancer biology. This review provides insights into targeted therapies and immunotherapies that have emerged using in vitro models and microbiome analysis. It focuses on therapeutic development using cell lines and the limitations of tumor heterogeneity and microenvironment. We explore the evolving landscape of breast cancer cell lines from two-dimensional (2-D) cultures to patient-derived xenograft (PDX) models advancing both fundamental and translational research. Patient-derived xenografts, cell line-derived xenografts (CDX), three-dimensional (3-D) cultures, organoids, and circulating tumor cells (CTC) models provide promising alternatives that capture the intricacies of the tumor microenvironment. This review bridges the gap between traditional cell lines and newer developments exploring the therapeutic and diagnostic advancements and needs for cell lines to expedite the progress in breast cancer research and treatment.

A Precise and Sustainable Doxycycline-Inducible Cell Line Development Platform for Reliable Mammalian Cell Engineering with Gain-of-Function Mutations.

For mammalian synthetic biology research, multiple orthogonal and tunable gene expression systems have been developed, among which the tetracycline (Tet)-inducible system is a key tool for gain-of-function mutations. Precise and long-lasting regulation of genetic circuits is necessary for the effective use of these systems in genetically engineered stable cell lines. However, current cell line development strategies, which depend on either random or site-specific integration along with antibiotic selection, are unpredictable and unsustainable, limiting their widespread use. To overcome these issues, we aimed to establish a Robust Overexpression via Site-specific integration of Effector (ROSE) system, a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated streamlined Tet-On3G-inducible master cell line (MCL) development platform. ROSE MCLs equipped with a landing pad facilitated the transcriptional regulation of various effector genes via recombinase-mediated cassette exchange. Long-term investigation revealed that the modular design of genetic payloads and integration sites significantly affected the induction capacity and stability, with ROSE MCLs exhibiting exceptional induction performance. To demonstrate the versatility of our platform, we explored its efficiency for the precise regulation of selection stringency, manufacturing of therapeutic antibodies with tunable expression levels and timing, and transcription factor engineering. Overall, this study demonstrated the effectiveness and reliability of the ROSE platform, highlighting its potential for various biological and biotechnological applications.

Establishment of a semi-continuous scale-down clone screening model for intensified perfusion culture.

PURPOSE: Perfusion cultures have been extensively used in the biotechnology industry to achieve high yields of recombinant products, especially those with stability issue. The WuXiUP™ platform represents a novel intensified perfusion that can achieve ultra-high productivity. This study describes a representative scale-down 24-deep well plate (24-DWP) cell culture model for intensified perfusion clone screening. METHODS: Clonal cell lines were expanded and evaluated in 24-DWP semi-continuous culture. Cell were sampled and counted daily with the aid of an automated liquid handler and high-throughput cell counter. To mimic perfusion culture, 24-DWP plates were spun down and resuspended with fresh medium daily. Top clones were ranked based on growth profiles and productivities. The best performing clones were evaluated on bioreactors. RESULTS: The selected clones achieved volumetric productivity (Pv) up to 5 g/L/day when expressing a monoclonal antibody, with the accumulative harvest Pv exceeding 60 g/L in a 21-day cell culture. Product quality attributes of clones cultured in 24-DWP were comparable with those from bioreactors. A high seeding strategy further shortened the clone screening timeline. CONCLUSION: In this study, a 24-DWP semi-continuous scale-down model was successfully developed to screen for cell lines suitable for intensified perfusion culture.

Transcriptomics and cell painting analysis reveals molecular and morphological features associated with fed-batch production performance in CHO recombinant clones.

Stable, highly productive mammalian cells are critical for manufacturing affordable and effective biological medicines. Establishing a rational design of optimal biotherapeutic expression systems requires understanding how cells support the high demand for efficient biologics production. To that end, we performed transcriptomics and high-throughput imaging studies to identify putative genes and morphological features that underpin differences in antibody productivity among clones from a Chinese hamster ovary cell line. During log phase growth, we found that the expression of genes involved in biological processes related to cellular morphology varied significantly between clones with high specific productivity (qP > 35 pg/cell/day) and low specific productivity (qP < 20 pg/cell/day). At Day 10 of a fed-batch production run, near peak viable cell density, differences in gene expression related to metabolism, epigenetic regulation, and proliferation became prominent. Furthermore, we identified a subset of genes whose expression predicted overall productivity, including glutathione synthetase (Gss) and lactate dehydrogenase A (LDHA). Finally, we demonstrated the feasibility of cell painting coupled with high-throughput imaging to assess the morphological properties of intracellular organelles in relation to growth and productivity in fed-batch production. Our efforts lay the groundwork for systematic elucidation of clone performance using a multiomics approach that can guide future process design strategies.

CRISPR-interceded CHO cell line development approaches.

For industrial production of recombinant protein biopharmaceuticals, Chinese hamster ovary (CHO) cells represent the most widely adopted host cell system, owing to their capacity to produce high-quality biologics with human-like posttranslational modifications. As opposed to random integration, targeted genome editing in genomic safe harbor sites has offered CHO cell line engineering a new perspective, ensuring production consistency in long-term culture and high biotherapeutic expression levels. Corresponding the remarkable advancements in knowledge of CRISPR-Cas systems, the use of CRISPR-Cas technology along with the donor design strategies has been pushed into increasing novel scenarios in cell line engineering, allowing scientists to modify mammalian genomes such as CHO cell line quickly, readily, and efficiently. Depending on the strategies and production requirements, the gene of interest can also be incorporated at single or multiple loci. This review will give a gist of all the most fundamental recent advancements in CHO cell line development, such as different cell line engineering approaches along with donor design strategies for targeted integration of the desired construct into genomic hot spots, which could ultimately lead to the fast-track product development process with consistent, improved product yield and quality.

High-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors.

Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all-in-one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site-specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy by 2.3-8.3-fold and 19-22-fold, respectively, compared to standard circular donors. Notably, Cas9-mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low-fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%-55% efficiency at two loci simultaneously in selected clones. The CRISPR-based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications.

Towards a scalable bioprocess for rAAV production using a HeLa stable cell line.

The majority of recombinant adeno-associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale-up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa-based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3-based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3-4 months, and presented rAAV2 volumetric production (bulk) > 3 × 1011 vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity-based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time-efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 1011 vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines.

Efficient site-specific integration in CHO-K1 cells using CRISPR/Cas9-modified donors.

BACKGROUND: Conventional methods applied to develop recombinant CHO (rCHO) cell line as a predominant host for mammalian protein expression are limited to random integration approaches, which can prolong the process of getting the desired clones for months. CRISPR/Cas9 could be an alternative by mediating site-specific integration into transcriptionally active hot spots, promoting homogenous clones, and shortening the clonal selection process. However, applying this approach for the rCHO cell line development depends on an acceptable integration rate and robust sites for the sustained expression. METHODS AND RESULTS: In this study, we aimed at improving the rate of GFP reporter integration to the Chromosome 3 (Chr3) pseudo-attP site of the CHO-K1 genome via two strategies; these include the PCR-based donor linearization and increasing local concentration of donor in the vicinity of DSB site by applying the monomeric streptavidin (mSA)-biotin tethering approach. According to the results, compared to the conventional CRISPR-mediated targeting, donor linearization and tethering methods exhibited 1.6- and 2.4-fold improvement in knock-in efficiency; among on-target clones, 84% and 73% were determined to be single copy by the quantitative PCR, respectively. Finally, to evaluate the expression level of the targeted integration, the expression cassette of hrsACE2 as a secretory protein was targeted to the Chr3 pseudo-attP site by applying the established tethering method. The generated cell pool reached 2-fold productivity, as compared to the random integration cell line. CONCLUSION: Our study suggested reliable strategies for enhancing the CRISPR-mediated integration, introducing Chr3 pseudo-attP site as a potential candidate for the sustained transgene expression, which might be applied to promote the rCHO cell line development.

Targeted integration in CHO cells using CRIS-PITCh/Bxb1 recombinase-mediated cassette exchange hybrid system.

Recombinant Chinese hamster ovary (CHO) cell line development for complex biotherapeutic production is conventionally based on the random integration (RI) approach. Due to the lack of control over the integration site and copy number, RI-generated cell pools are always coupled with rigorous screening to find clones that satisfy requirements for production titers, quality, and stability. Targeted integration into a well-defined genomic site has been suggested as a possible strategy to mitigate the drawbacks associated with RI. In this work, we employed the CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) system in combination with the Bxb1 recombinase-mediated cassette exchange (RMCE) system to generate an isogenic transgene-expressing cell line. We successfully utilized the CRIS-PITCh system to target a 2.6 kb Bxb1 landing pad with homology arms as short as 30 bp into the upstream region of the S100A gene cluster, achieving a targeting efficiency of 10.4%. The platform cell line (PCL) with a single copy of the landing pad was then employed for the Bxb1-mediated landing pad exchange with an EGFP encoding cassette to prove its functionality. Finally, to accomplish the main goal of our cell line development method, the PCL was applied for the expression of a secretory glycoprotein, human recombinant soluble angiotensin-converting enzyme 2 (hrsACE2). Taken together, on-target, single-copy, and stable expression of the transgene over long-term cultivation demonstrated our CRIS-PITCh/RMCE hybrid approach might possibly improve the cell line development process in terms of timeline, specificity, and stability. KEY POINTS: • CRIS-PITCh system is an efficient method for single copy targeted integration of the landing pad and generation of platform cell line • Upstream region of the S100A gene cluster of CHO-K1 is retargetable by recombinase-mediated cassette exchange (RMCE) approach and provides a stable expression of the transgene • CRIS-PITCh/Bxb1 RMCE hybrid system has the potential to overcome some limitations of the random integration approach and accelerate the cell line development timeline.

CRISPR Technologies in Chinese Hamster Ovary Cell Line Engineering.

The Chinese hamster ovary (CHO) cell line is a well-established platform for the production of biopharmaceuticals due to its ability to express complex therapeutic proteins with human-like glycopatterns in high amounts. The advent of CRISPR technology has opened up new avenues for the engineering of CHO cell lines for improved protein production and enhanced product quality. This review summarizes recent advances in the application of CRISPR technology for CHO cell line engineering with a particular focus on glycosylation modulation, productivity enhancement, tackling adventitious agents, elimination of problematic host cell proteins, development of antibiotic-free selection systems, site-specific transgene integration, and CRISPR-mediated gene activation and repression. The review highlights the potential of CRISPR technology in CHO cell line genome editing and epigenetic engineering for the more efficient and cost-effective development of biopharmaceuticals while ensuring the safety and quality of the final product.

Perfusion culture of Chinese Hamster Ovary cells for bioprocessing applications.

Much of the biopharmaceutical industry's success over the past 30 years has relied on products derived from Chinese Hamster Ovary (CHO) cell lines. During this time, improvements in mammalian cell cultures have come from cell line development and process optimization suited for large-scale fed-batch processes. Originally developed for high cell densities and sensitive products, perfusion processes have a long history. Driven by high volumetric titers and a small footprint, perfusion-based bioprocess research has regained an interest from academia and industry. The recent pandemic has further highlighted the need for such intensified biomanufacturing options. In this review, we outline the technical history of research in this field as it applies to biologics production in CHO cells. We demonstrate a number of emerging trends in the literature and corroborate these with underlying drivers in the commercial space. From these trends, we speculate that the future of perfusion bioprocesses is bright and that the fields of media optimization, continuous processing, and cell line engineering hold the greatest potential. Aligning in its continuous setup with the demands for Industry 4.0, perfusion biomanufacturing is likely to be a hot topic in the years to come.

Rapidly accelerated development of neutralizing COVID-19 antibodies by reducing cell line and CMC development timelines.

Since the Coronavirus Disease 2019 (COVID-19) outbreak, unconventional cell line development (CLD) strategies have been taken to enable development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing antibodies at expedited speed. We previously reported a novel chemistry, manufacturing, and control (CMC) workflow and demonstrated a much-shortened timeline of 3-6 months from DNA to investigational new drug (IND) application. Hereafter, we have incorporated this CMC strategy for many SARS-CoV-2-neutralizing antibody programs at WuXi Biologics. In this paper, we summarize the accelerated development of a total of seven antibody programs, some of which have received emergency use authorization  approval in less than 2 years. Stable pools generated under good manufacturing practice (GMP) conditions consistently exhibited similar productivity and product quality at different scales and batches, enabling rapid initiation of phase I clinical trials. Clones with comparable product quality as parental pools were subsequently screened and selected for late-stage development and manufacturing. Moreover, a preliminary stability study plan was devised to greatly reduce the time required for final clone determination and next-generation sequencing-based viral testing was implemented to support rapid conditional release of the master cell bank for GMP production. The successful execution of these COVID-19 programs relies on our robust, fit for purpose, and continuously improving CLD platform. The speed achieved for pandemic-related biologics development may innovate typical biologics development timelines and become a new standard in the industry.

Structural analysis of random transgene integration in CHO manufacturing cell lines by targeted sequencing.

Genetically modified CHO cell lines are traditionally used for the production of biopharmaceuticals. However, an in-depth molecular understanding of the mechanism and exact position of transgene integration into the genome of pharmaceutical manufacturing cell lines is still scarce. Next-generation sequencing (NGS) holds great promise for strongly facilitating the understanding of CHO cell factories, as it has matured to a powerful and affordable technology for cellular genotype analysis. Targeted Locus Amplification (TLA) combined with NGS allows for robust detection of genomic positions of transgene integration and structural genomic changes occurring upon stable integration of expression vectors. TLA was applied to generate comparative genomic fingerprints of several CHO production cell lines expressing different monoclonal antibodies. Moreover, high producers resulting from an additional round of transfection of an existing cell line (supertransfection) were analyzed to investigate the integrity and the number of integration sites. Our analyses enabled detailed genetic characterization of the integration regions with respect to the number of integrates and structural changes of the host cell's genome. Single integration sites per clone with concatenated transgene copies could be detected and were in some cases found to be associated with genomic rearrangements, deletions or translocations. Supertransfection resulted in an increase in titer associated with an additional integration site per clone. Based on the TLA fingerprints, CHO cell lines originating from the same mother clone could clearly be distinguished. Interestingly, two CHO cell lines originating from the same mother clone were shown to differ genetically and phenotypically despite their identical TLA fingerprints. Taken together, TLA provides an accurate genetic characterization with respect to transgene integration sites compared with conventional methods and represents a valuable tool for a comprehensive evaluation of CHO production clones early in cell line development.

Rapid antibody conformational screening by matrix-assisted laser desorption/ionization hydrogen-deuterium exchange mass spectrometry.

Recent advances in the field of cancer biology have accelerated the discovery and development of novel biopharmaceuticals. At the forefront of these drug development efforts are high-throughput screening, compressed timelines, and limited sample quantities, all characteristic of the discovery space. To meet program targets, large numbers of protein variants must be produced, screened, and characterized, presenting a daunting analytical challenge. Additionally, the higher-order structure is paramount for protein function and must be monitored as a critical quality attribute. Matrix-assisted laser desorption/ionization mass spectrometry has been utilized as an ultra-fast, automatable, sample-sparing analytical tool for biomolecules. Our group has published applications integrating hydrogen-deuterium exchange mass spectrometry with matrix-assisted laser desorption/ionization mass spectrometry for the rapid conformational characterization of small proteins, the current work expands this application to monoclonal and bi-specific antibodies. This study demonstrates the ability of the methodology, matrix-assisted laser desorption/ionization hydrogen-deuterium exchange mass spectrometry, to detect conformational differences between bi-specific antibodies from different expression hosts. These conformational differences were validated by orthogonal techniques including circular dichroism, nuclear magnetic resonance, and size-exclusion chromatography hydrogen-deuterium exchange mass spectrometry. This work demonstrates the utility of applying the developed methodology as a rapid conformational screening tool to triage samples for further analytical characterization.

High efficiency sorting and outgrowth for single-cell cloning of mammalian cell lines.

Single-cell selection and cloning is required for multiple bioprocessing and cell engineering workflows. Dispensing efficiency and outgrowth were optimized for multiple common suspension (CHO ES, Expi293F, and Jurkat) and adherent (MCF-7, A549, CHO-K1, and HEK293) cell lines. Single-cell sorting using a low pressure microfluidic cell sorter, the WOLF Cell Sorter, was compared with limiting dilution at 0.5 cells/well to demonstrate the increased efficiency of using flow cytometry selection of cells. In this work, there was an average single cell deposition on Day 0 of 89.1% across all the cell lines tested compared to 41.2% when using limiting dilution. After growth for 14 days, 66.7% of single-cell clones sorted with the WOLF Cell Sorter survived and only 23.8% when using limiting dilution. Using the WOLF Cell Sorter for cell line development results in higher viable single-cell colonies and the ability to select subpopulations of single-cells using multiple parameters.

Recent developments in miRNA based recombinant protein expression in CHO.

It is widely accepted that the growing demand for recombinant therapeutic proteins has led to the expansion of the biopharmaceutical industry and the development of strategies to increase recombinant protein production in mammalian cell lines such as SP2/0 HEK and particularly Chinese hamster ovary cells. For a long time now, most investigations have been focused on increasing host cell productivity using genetic manipulating of cellular processes like cell cycle, apoptosis, cell growth, protein secretory and other pathways. In recent decades MicroRNAs beside different genetic engineering tools (e.g., TALEN, ZFN, and Crisper/Cas) have attracted further attention as a tool in the genetic engineering of host cells to increase protein expression levels. Their ability to simultaneously target multiple mRNAs involved in one or more cellular processes made them a favorable tool in this field. Accordingly, this study aimed to review the methods of selecting target miRNA for cell line engineering, miRNA gain- or loss-of-function strategies, examples of laboratory and pilot studies in this field and discussed advantages and disadvantages of this technology.

Accelerating and de-risking CMC development with transposon-derived manufacturing cell lines.

The development of highly productive, genetically stable manufacturing cell lines is on the critical path to IND filing for protein-based biologic drugs. Here, we describe the Leap-In Transposase® platform, a novel transposon-based mammalian (e.g., Chinese hamster ovary) cell line development system that produces high-titer stable pools with productivity and product quality attributes that are highly comparable to clones that are subsequently derived therefrom. The productivity distributions of clones are strongly biased toward high producers, and genetic and expression stability is consistently high. By avoiding the poor integration rates, concatemer formation, detrimental transgene recombination, low average expression level, unpredictable product quality, and inconsistent genetic stability characteristic of nonhomologous recombination methods, Leap-In provides several opportunities to de-risk programs early and reduce timelines and resources.

The use of site-specific recombination and cassette exchange technologies for monoclonal antibody production in Chinese Hamster ovary cells: retrospective analysis and future directions.

Chinese hamster ovary (CHO) cell-based platforms are the most widely used for the biomanufacturing of complex therapeutic proteins, such as monoclonal antibodies (mAbs). The development of high-producing clones that are stable and amenable to large-scale cultures is essential to advance a molecule toward clinical evaluation. Nevertheless, the generation of such clones generally relies on random integration of an expression plasmid encoding the therapeutic protein gene into the host genome. The ensuing clone selection relying on empirical screens and cell line characterization is extensive and time-consuming. An emerging paradigm in CHO cell line development is the use of site-specific recombinases to enable the integration of therapeutic transgenes into pre-marked chromosomal locations with defined expression characteristics. Recombinase-mediated cassette exchange (RMCE) provides a sophisticated alternative to conventional CHO cell line development, leading to the generation of more consistent and reliable clones and may ultimately shorten the "time-to-clinic" of recombinant therapeutics. Herein, we review the recent advances in the use of site-specific recombination systems and their associated cassette exchange technologies for the rapid generation of stable CHO clones with predictable growth, stability, quality and productivity characteristics. Particular emphasis is placed on cassette exchange technologies currently used in the industry. We also discuss the technical hurdles associated with uses of site-specific recombinase systems in CHO cells, illustrate how these problems can be mitigated and provide a perspective on future work concerning these systems.

Random epigenetic modulation of CHO cells by repeated knockdown of DNA methyltransferases increases population diversity and enables sorting of cells with higher production capacities.

Chinese hamster ovary (CHO) cells produce a large share of today's biopharmaceuticals. Still, the generation of satisfactory producer cell lines is a tedious undertaking. Recently, it was found that CHO cells, when exposed to new environmental conditions, modify their epigenome, suggesting that cells adapt their gene expression pattern to handle new challenges. The major aim of the present study was to employ artificially induced, random changes in the DNA-methylation pattern of CHO cells to diversify cell populations and consequently increase the finding of cell lines with improved cellular characteristics. To achieve this, DNA methyltransferases and/or the ten-eleven translocation enzymes were downregulated by RNA interference over a time span of ∼16 days. Methylation analysis of the resulting cell pools revealed that the knockdown of DNA methyltransferases was highly effective in randomly demethylating the genome. The same approach, when applied to stable CHO producer cells resulted in (a) an increased productivity diversity in the cell population, and (b) a higher number of outliers within the population, which resulted in higher specific productivity and titer in the sorted cells. These findings suggest that epigenetics play a previously underestimated, but actually important role in defining the overall cellular behavior of production clones.

Genome-wide analysis of single nucleotide variants allows for robust and accurate assessment of clonal derivation in cell lines used to produce biologics.

A clonally derived (or "monoclonal") cell line is a cell population derived from a single progenitor cell. Clonally derived cell lines are required for many biotechnological applications. For instance, recombinant mammalian cells used to produce therapeutic proteins are expected by regulatory authorities to be clonally derived. Assurance of clonal derivation (or "clonality") is usually obtained from the characterization of the procedure used for cell cloning, for instance by assessing the success rate of single-cell sorting but not by assessing the cell line itself. We have developed a method to assess clonal derivation directly from the genetic makeup of cells. The genomic test of clonality is based on whole-genome sequencing and statistical analysis of single nucleotide variants. This approach quantifies the clonal fractions present in nonclonal samples and it provides a measure of the probability that a cell line is derived from a single cell. Upon experimental validation of the test, we show that it is highly accurate and that it can robustly detect minor clonal fractions of as little as 1% of the cell population. Moreover, we find that it is applicable to various cell line development protocols. This approach can simplify development protocols and shorten timelines while ensuring clonal derivation with high confidence.