Cell Line Development With Intelligent Tools, Services, and Solutions for Your Unique Needs

Usually both, toxicology and first-in-human clinical materials are generated using the same clonal-derived cells to ensure safety and minimize any development risks. However, cell line development with single cell cloning is time consuming and aggravated by the time needed to screen for a lead clone based on cell line stability and manufacturability. In order to achieve faster timelines, there are approaches using pools of several clones for earlier production of drug substance for regulatory filing-enabling toxicology studies, and later the final single clone will be selected for production of clinical materials.

Post-translational modification is a common event during protein production from eukaryotic cells. It includes a wide variety of chemical modifications, such as acetylation, amidation, carboxylation, phosphorylation, and glycosylation. These important chemical modifications are essential for protein folding, stability, and function. 

Among these various post-translational modifications, glycosylation is the most complex one from the perspective of chemical heterogeneity of modifications because it involves the covalent attachment of sugar moieties to proteins. There are two common types of glycosylation: O-linked glycosylation and N-linked glycosylation.

The glycosylation pattern plays an important role in the biological activity of a protein, such as that of afucosylated monoclonal antibodies (mAbs).

During therapeutic antibody production via mammalian cells, such as Chinese hamster ovary (CHO) cells, N-linked glycosylation is commonly observed. For the IgG1 type of antibodies, there is a conserved site in the heavy chain Fc region for glycosylation. During the post-translational modification, multiple sugar moieties (e.g., N-acetylglucosamine (GlcNAc), mannose, fucose, galactose) can be assembled with complex combinations to form a variety of glycosylation patterns. Advances have been made primarily in upstream processing, including mammalian cell line engineering, to yield more predictably glycosylated mAbs and the addition of media supplements during fermentation to manipulate the metabolic pathways involved in glycosylation. Another strategy is the implementation of novel downstream technologies, such as the use of Fcγ-based affinity ligands for the separation of mAb glycovariants.
 

It is common in cell line development to get a certain percentage of clones that unstable, meaning that they lose productivity over time due to a loss of gene copies or gene silencing effects. Depending on the applied technology the proportion of unstable clones can be up to 50 % or higher. However, with mature technologies such as Sartorius’ Cellca Cell Line Development Technology the percentage of stable clones can be  80 % or more.  

Nearly all cellular systems are heterogeneous, contributing to specialized functionality and improved survival. A profound comprehension of single cell heterogeneity on genomic, epigenomic, transcriptomic and proteomic level is critical for understanding its impact on the functioning of organism in both healthy and diseased condition.

Most of our current knowledge about different cell and tissue types comes from bulk assays though, analyzing hundreds to millions of cells together, which may highly underestimate the true spectrum of cellular heterogeneity.

Modern technologies allow the analysis on single cell level therefore taking a huge step forward in the understanding of cellular heterogeneity and its implications:

• Single cell heterogeneity analysis can help to clarify developmental pathways, e.g., how stem cells make their fate decision, contributing to the development and maintenance of differentiated tissue. This is not only important for understanding malignancies caused by missing or misdirected differentiation but especially for the field of regenerative medicine.
• In order to analyze the cellular heterogeneity on single cell level e.g., by whole genome amplification (WGA), SNP detection or sequencing target cells have to be selected and isolated in high purity from a heterogeneous background. The CellCelector™ is the perfect tool for the automated identification and isolation of 100 % pure individual single cells offering many advantages over other methods.
• Target cells are detected and selected for isolation based on microscopic imaging. This enables target cell selection utilizing fluorescent markers but also through distinct morphological features a target cell might express and that is visible in microscopy. This allows label free detection of target cells independently of fluorescent markers. Furthermore, a live image during isolation as well as automated recording of before and after picking images of each isolated single cell create a comprehensive documentation of the picking process ensuring that only target cells have been picked without contamination of non-target cells. The fast isolation process is extremely gentle resulting in high cell integrity (up to 100 % outgrowth rate in cloning applications) and outstanding transfer efficiency of up to 100%.