Verified in-situ plate seeding: a new frontier in cell line development | Advanced Instrument

Verified in-situ plate seeding: a new frontier in cell line development

Verified In-Situ Plate Seeding (VIPS) takes single cell cloning and image based clonality assurance to a whole new level of quality.

Forty years ago, in 1982, the first recombinant DNA product was approved by the FDA, thus marking the beginning of the modern biopharmaceutical industry. Since then, the number of approved biologics has grown1. Currently, more than 350 biologics are commercially present on the market, and their share is further expanding. As Boston, Massachusetts’ BCC Research reported in November 2021, the global market for biologic therapeutic drugs should increase from $285.5 billion in 2020 to $421.8 billion by 2025, a compound annual growth rate of 8.1% during the forecast period of 2020–20252. Factors such as the demographic shift toward an older population, the correlated rise of chronic diseases and the successful use of biologics in the prevention and treatment of many diseases such as cancer have contributed to the constant growth of the biopharmaceutical market.

Cell line technologies: improving speed and efficiency in biologics development

In order to keep up with this exponential market development, biopharmaceutical companies are looking for innovative technologies allowing them to increase the efficiency of their workflows and to optimize product development processes3. As biologics are products made in living organisms4, the development of stable cell lines and master cell banks (MCB) needed for their manufacturing is of utmost importance for both manufacturers and regulators. In fact, to ensure product quality and consistency and to meet regulatory requirements, companies are required to prove that their Master Cell Banks (MCBs) are of clonal origin, i.e. derived from a single cell. However, the cell line development (CLD) process, which involves conventional methodologies, is often an inefficient, tedious and time-consuming process that shows many limitations and challenges.

“The traditional method for establishing clonally derived cell lines is limiting dilution (LD),” explains Marta Rucka, Global Product Leader at Advanced Instruments. “However, LD is extremely time consuming and is prone to human error as clonality assessment is either confirmed by visual inspection using a microscope or is based on statistical probability of achieving a single cell in a well.” Automated cell seeders such as Solentim VIPS™ from Advanced Instruments help overcome these challenges as they allow much higher seeding efficiency, drastically reducing the rate of human error and significantly speeding up the entire process.

Double lock of assurance: combining high efficiency single cell seeding with image-based clonality assurance

“VIPS is an automated single cell seeder used in cell line development,” remarks Rucka. “However, it’s not just a simple depositing device that allows scientists to seed individual cells. It also supplies image-based proof of clonality.” In fact, immediately after dispensing a droplet containing an individual cell into an empty uncoated well, VIPS verifies the existence of a single cell in the well by taking high-quality images of the 30 nanoliter droplet. The VIPS cell detection software applies artificial intelligence to identify the presence of a single or multiple cells in the well. If no cell is detected, the process is repeated up to 16 times within the same well until a single cell is detected. The 20 layer z-stack image of the droplet is the first stage in providing image-based evidence of clonality. Immediately after single cell detection within the droplet, the well is automatically filled with a growth medium. Subsequently, a whole well picture is taken to provide further assurance of single cell seeding. Following the double lock assurance on day 0, VIPS can daily take whole-well images, allowing the monitoring and documentation of colony outgrowth over a desired period of time.

“I think the beauty of this technology is that it gives customers the chance to visualize the single cell dispensing and cell detection progress in real time. The results are displayed on screen and customers can make any adjustments early in the process if required.”
Marta Rucka, Global Product Leader at Advanced Instruments

A double lock of clonality assurance helps to fulfill regulatory requirements and support Investigational New Drug (IND) applications; it also provides significant advantages over traditional labor-intensive methods of single cell cloning such as limiting dilution.

Proof of clonality: a crucial regulatory requirement in the manufacturing of biologics

Assurance of clonal derivation is a crucial component of quality and consistency of MCB used for the manufacturing of therapeutic antibodies, viral vectors and highly valuable human-induced pluripotent stem cells (iPSCs). Human iPSCs are adult stem cells which have been genetically reprogrammed to an embryonic stem cell-like state and can be triggered to differentiate into, for example, hepatocytes, neurons or skeletal myocytes5. Regulatory agencies all over the world, including the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), seek assurance of clonally-derived MCB in regulatory submissions.

White Paper
Assurance of Clonality: a key regulatory requirement for biotherapeutics

Ensuring clonal origin of production cell lines is a critical step in the manufacturing of advanced biotherapeutics. Innovative devices help increase the efficiency of the cloning process and bring image-based assurance of clonality.

“We have different customers operating in the cell line development area,” explains Rucka. “Whether they produce therapeutic antibodies, viral vectors or work with stem cells, monoclonality is absolutely essential to them. In particular, the use of iPSCs in drug discovery, synthetic biology and cell therapy is rapidly gaining attention.” In fact, due to their pluripotency, iPSCs offer a unique platform to model human diseases and can potentially be manipulated to fix disease-causing defects. Their huge potential makes them a game changer in cell therapy.

Also in the context of iPSC, monoclonality is absolutely crucial to ensure that the cell population is homogeneous in terms of genotype, karyotype, morphology and functionality. However, current techniques such as LD, colony picking and fluorescence-activated cell sorting (FACS) are time consuming and expensive; these techniques are also incompatible with the sensitivity of iPSCs and provide insufficient evidence of clonality. Moreover, they do not allow a robust manipulation of iPSCs as single cells, which thus poses a significant biological and technical hurdle.

“Scientists working with iPSCs want to edit them, give them a new identity and safely administer them to patients,” summarizes Rucka. ”Hence, it is absolutely crucial to be able to control these processes and bring evidence of clonality to the regulatory bodies.” Thanks to its double lock of assurance of clonality, VIPS can provide the required evidence, which is supported by extremely high quality images. Furthermore, Advanced Instruments developed iPSC confluency software, which employs an artificial intelligence (AI)-driven algorithm designed to accurately recognize and image the morphology of iPSCs, to provide more information about clonal outgrowth and cell proliferation. In fact, the combination of AI, automation and the highest quality imaging supports biopharmaceutical companies aiming to establish standardized processes for the generation of clonal MCBs.

Moving beyond the limits of traditional methods: enhancing productivity and reducing timelines

The high seeding efficiency of VIPS allows scientists to consistently speed up the CLD process, while cutting down on the plastic and other consumables. This significant improvement of the single cell seeding step helps to reduce the time and number of plates required by limiting dilutions, and it achieves better results. “Even though traditional methods are still accepted and used in the manufacturing of biologics, many customers are looking at innovative processes,” explains Rucka. ”Automation grants higher efficiency and results, with significant time and money savings and a more sustainable workflow. Furthermore, it eliminates the bias that comes with assessing clonality by the human eye.” Speeding up product development timelines while guaranteeing high quality and safety of the end product is a priority considering that many innovative biologics are designed to address unmet medical needs6.

Case Study
Successfully filing an IND using Solentim’s VIPS™ single cell cloning system

“The VIPS™ system and software has helped our company to deliver a high-quality data package that meets global regulatory standards. The ease of use and reliability of the VIPS freed up valuable time and resources to help with platform development and to support the discovery team for other R&D activities.”
Andy Tsun, PhD, Co-Founder and VP of Discovery Biology, Biotheus Inc.

A case study conducted by the CLD group at Janssen R&D (Spring House, Pennsylvania, USA) showed that moving to the VIPS platform made a significant difference in terms of seeding efficiency, incidence of ghost wells, cell survival and cell outgrowth. Specifically, the study found that:

  • VIPS had seeding efficiencies as high as 87%, even at the lowest cell density, and correctly identified wells containing a cell 95% of the time.
  • The incidence of ghost wells (false negatives) could be significantly reduced (0,06% compared to 10% using limiting dilution).
  • VIPS allowed seeding and confirmed single cells in most of the wells on each plate.
  • Compared to manual LD, VIPS’ gentle dispensing method achieved a similar outgrowth rate or cloning efficiency (31.9%), but doubled the number of colonies per plate.
  • The single-step VIPS technology more than halved the time for single cell cloning workflows compared to a typical two step LD methodology.

“We now can screen more clones in far fewer plates, which is great because it lessens the workload in terms of the number of plates you have to handle,” explains Tom
Kelly, scientist in the CLD group at Janssen.

Case Study
Doubling the Speed of Cell Line Development in a Large Pharmaceutical Company

For the past 10 years, the Cell Line Development (CLD) group at Janssen R&D (Spring House, PA) had used the same two-step process to develop the final cell cultures in order to manufacture new biologic drugs. Thanks to Solentim’s VIPS™ technology, they’ve reduced that process to single step – halving development times while still satisfying the clonality requirements of the medicines’ regulators.

Solentim VIPS and Cell Metric: a powerful combination

Advanced Instruments’ Cell Metric® is a high contrast imager which captures and records a whole well image of the single cell at day 0 and every day after in order to identify and characterize clonal outgrowth. The instrument also creates a clonality report. VIPS and Cell Metric are designed to be fully compatible and to seamlessly integrate with each other to optimize and simplify the workflow. “Cell Metric is the market leader for monitoring clonal outgrowth,” remarks Rucka. ”It takes extremely high-quality images. The use of both VIPS and Cell Metric together allows customers to monitor the whole clonal outgrowth process, starting from a single cell.” Data-sync between the two instruments enables sharing of seeding data and images, day 0 evidence of clonality, whole well images and subsequent outgrowth time point images. Moreover, the integration of VIPS and Cell Metric in existing workflows is straightforward. Cell Metric is compatible with a number of different automated platforms and works with different robotic arms. As well, depending on the level of automation required, software and features can be tailored to individual needs.

A customer-centric approach: meeting the needs of biopharmaceutical companies

Customer feedback plays a pivotal role in product development at Advanced Instruments, Rucka stresses. “We regularly evaluate customer reviews. This exercise allows us to gather feedback, uncover any unmet requirements and evaluate customer experiences, “ explains Rucka. “We interview different scientists, list their needs and top requirements, evaluating the feasibility of any potential new features.” Implementing customer requirements and feedback drives the product development roadmap at Advanced Instruments. This customer-centric approach enables the implementation of effective solutions to improve CLD workflow efficiency. “We strive to make a difference for our customers in the extremely competitive biopharmaceutical market,” concludes Rucka.