Successful cell line development is a critical factor for achieving effective biomanufacturing. As a result, it is a core component of modern cell-based bioprocessing. However, cell line development still poses remarkable challenges for biopharmaceutical companies striving to quickly bring new biologics to the market. In fact, the development of stable cell lines represents a main rate-limiting step during the initial phases of biotherapeutics development. Overall, establishing robust and consistent workflows is a prerequisite for guaranteeing product quality, being in compliance with regulatory requirements and conducting best practices in biotherapeutic manufacturing.
As described in the 2020 FDA Chemistry Manufacturing and Controls (CMC) Guidelines and the Investigational New Drug (IND) Application for cell and gene therapy, both regulators and manufacturers are aiming at standardizing best practices around bioprocessing workflows1. This is essential since cell lines used to manufacture biopharmaceuticals need to fulfill strict requirements, which include that the cell lines need to be of clonal origin, i.e. derived from one single cell, and are to be grown in the absence of animal products.
In addition, when laboratories succeed in developing an efficient cell line selection process, they may still struggle in determining the best conditions possible for successful single cell outgrowth into colonies.
Cloning efficiency: establishing the best conditions for single cells to grow and proliferate into colonies
Mammalian cells used in biomanufacturing have quite a journey to take from transfection to cell banking: Along the way, they need to adapt to different conditions and environments. Following transfection, recovery and selection, pools of transfected cells are seeded as single cells and cultured in static conditions initially in either 96 or 384 well plates and then gradually expanded onto larger well formats (such as 24-well plates). Next, cells are moved to shaking flasks or spin tubes, and then finally banked down. Determining the best conditions possible for each step of this multifaceted process is challenging: Scientists need to optimize and harmonize many factors including the type of the host cell line, the clonal selection method, the timing of each stage, the plate type, and the media type and volume.
Among these many factors, careful selection of cell culture media and supplements is of utmost importance to alleviate cellular stress, support cell growth and maximize yields of recombinant proteins. In fact, the optimization of the culture media composition has a direct and positive impact on cellular outgrowth and, therefore, on workflow efficiency2.
“Determining the best conditions possible for each stage of cell line development is essential for guaranteeing a high cloning efficiency (the number of single cells that grow into a resultant colony) and hence a reduction of the time and labor spent processing the clones,” remarks Morris. “Advanced Instruments’ Insti Reagents are animal component-free cell culture supplements designed for supporting CHO and HEK cells throughout the entire process. They are composed of recombinant proteins, plant extracts and other chemical components to confer major growth and survival advantages within cell line development.”
InstiGRO™: Enhancing cell growth at the single cell level
Historically, cell lines are grown in the presence of animal-derived products, mainly fetal bovine or calf serum, to ensure proper cell growth. In fact, animal sera provide essential nutrients needed for cell growth, proliferation and differentiation3. However, the use of animal sera presents several concerns, mainly related to the risk to human health posed by the bovine spongiform encephalopathy (BSE). To overcome these issues, animal-free alternatives have been developed and tested for their ability to support long-term cell growth4.
However, optimizing and improving growth conditions in animal-free culture conditions is particularly challenging. In fact, serum-free conditions are suboptimal for the cells needing to recover after the stressful cloning process. High producing cells are especially sensitive to media conditions and are likely to experience a growth disadvantage compared to non-producers. In addition, a high number of single cells often do not survive the static cloning stage. “An alternative to animal sera is conditioned media,” says Morris. “Conditioned media is obtained from the cell culture before cloning and contains growth cytokines that basically fool the cells into thinking that they are not alone.” However, conditioned media show high variability, and it is not well defined; further, its success strongly depends on the health of the cultured cells before cloning.
To overcome the limits of both animal sera and conditioned media, Advanced Instruments’ InstiGRO is a proven, easy-to-use formulation. This supplement allows scientists to standardize the initial phases of the cell line development process and meet regulatory requirements. As an animal-free supplement, InstiGRO has been tailored to optimize single cell growth conditions and support early cell line development. “Formulations of InstiGRO exist for both HEK and CHO cells and support these cells during single cell isolation and the early growth phase in static conditions,” Morris says. “Their use accelerates the whole cell line development process by increasing the number of viable clonal colonies per plate.” In fact, InstiGRO allows dramatic improvement in the number of clonal cells that grow out of the early static stage of the cell line development process. Experiments show that supplementing cell culture media with InstiGRO can lead to a 30-fold increase in the number of clonal colonies compared to conditioned media.
Moreover, combining Advanced Instruments’ high-efficiency, single-cell seeder, VIPS™, and InstiGRO further enhances the efficiency of the cell line development process. “Our instruments work very nicely in tandem with the cell culture reagents, and the advantages of combining VIPS and InstiGRO for CHO and HEK are huge,” remarks Morris. “VIPS allows a much higher seeding efficiency and outgrowth than limiting dilution, and InstiGRO further enhances cell outgrowth.” In fact, internal data shows that the combination of VIPS and InstiGRO dramatically and consistently increases the percentage of clonal outgrowth, which can be closely monitored using the Cell Metric® whole well imager. Further, clones productivity can be assessed and documented using Advanced Instruments’ ICON™, a multifunctional instrument that enables the triage of clones via low volume titer and viable cell assays. “Selecting the top-producer clones is a crucial step in the biopharmaceutical workflow,” says Morris. “In fact, it is only those clones that are able to produce high enough quantities of recombinant proteins to guarantee efficient drug manufacturing.”
InstiSHAKE™: Supporting cell transition from static plates into suspension culture
Once single cells start growing into clonal colonies, they are transitioned through multiple static format cultures and eventually into shaking flasks to allow further expansion. This transfer from static to suspension is effectively a selection step for clones. “When you suddenly put cells, which have been in static growth conditions for a number of weeks, into shaking growth conditions, they undergo a shock and viability can drop, or even worse, the cell line can die completely,” remarks Morris. “Cells need time to recover and adapt. InstiSHAKE is designed to support cells during this transition stage, accelerating their adaptation process and boosting cell survival.” In fact, the transition from static plates to flasks is a key step toward the ultimate goal of scaling up to bioreactors, where the cells will be in high density suspension culture which is needed to ensure production of sufficient quantities of a biopharmaceutical. Moreover, fast cell proliferation, without compromising protein production, results in significant timeline shortening, therefore enhancing workflow efficiency.
InstiTHAW™: Optimizing clone preservation in cell banking
After the expansion stage, the newly developed cell lines need to be cryopreserved and stored until further use as Master Cell Banks (MCBs). The preservation of MCBs at low temperatures for an extended period of time is crucial for establishing an efficient biomanufacturing process5. Therefore, the development of strategies that ensure a quick recovery of highly viable and functional cells after thawing is essential. “InstiTHAW is designed to support cells undergoing both the freezing and the thawing process,” explains Morris. “It can be used as an additive in the freezing solution or to optimize cell recovery after revival.” In fact, this supplement helps keep cell loss as low as possible during the cryopreservation step and accelerates cell recovery and proliferation post-thawing. Enhancing post-thaw viability and allowing cells to move quickly from thawing to exponential growth can dramatically enhance process efficiency.
MatriClone™: single cell cloning matrix for stem cells
Due to their massive proliferation and differentiation potential, hiPSCs are increasingly used in autologous and allogeneic cell therapies. However, selecting and expanding stable and viable hiPSCs clones is a difficult task. The hiPSCs need cell culture matrices to support their growth into colonies. One of the most common approaches for clonal hiPSCs expansion is based on the pre-coating of the culture plates with a commercially available coating matrix, a gelatinous extract taken from mouse tumor cells that contains extracellular matrices. However, this matrix (derived from mouse tumors) is not animal-free, and the pre-coating process is time-consuming and difficult to standardize.
“Solentim MatriClone™ from Advanced Instruments is a soluble laminin matrix,” says Morris. “It is animal-free and can be mixed in solution with the cells prior and post clonal selection, eliminating the need for pre-coating. It is also superior for optical imaging of the clones in the 96 well cloning plates.” Mixing MatriClone directly with the cells guarantees a more homogeneous and even coating of the well bottom. Moreover, the combined use of the VIPS™ cell seeding platform and MatriClone for culture condition optimization allows a highly efficient and automated hiPSCs subcloning, improving clonal colony outgrowth and effectively reducing workflow time and costs. Further, cells maintain their viability, pluripotency, phenotype and genomic integrity during the post-cloning expansion phase. For regulatory purposes, cell line clonality is documented immediately after dispensing the single cells into the plate’s well, post media fill and throughout the entire colony expansion process. It should be noted that where customers use an alternative matrix, the cells can be adapted to the MatriClone for the single cell seeding steps, and then easily passed back into the media of choice post cloning.
Example plate overviews from VIPS software
Optimizing cell line development workflows, accelerating timelines, eliminating bottlenecks and supporting high-value cells throughout their development journey are the main factors influencing the efficiency and quality of biopharmaceutical manufacturing. From finding the optimal cell line to ensuring its clonal origin and consistently expanding the cells, a robust workflow is critical for successfully bringing a new biotherapeutic to the market. “Our cell culture supplements are designed to accelerate the cell line development process and make it more efficient,”concludes Morris. “Our customers do not need to change the media that they are using in any way. They just need to add the supplements to their outgrowth media in the appropriate concentration to greatly enhance cellular outgrowth.”