Glycosylation is an important factor for the efficacy and safety of many biopharmaceuticals. Cell lines differ vastly in their glycosylation potential, and assessing, as well as engineering, glycosylation is gaining importance in the early development of biopharmaceuticals. A range of mass spectrometric methods will be presented for the facile, high-resolution assessment of glycosylation at the cellular and molecular levels to support early development and clone selection.

It can be a critical decision by any institution to select right host cell line; it can impact quality, cost of goods and timeline. The presentation describes development studies of S-CHOice, Samsung’s proprietary cell line, which can help achieve high quality product, adding value to Samsung’s one-stop CDMO capability.

Correct assembly of subunits in multi-specific antibody therapeutics is a critical quality attribute (CQA) with direct impact on biological activity. Here, we describe a high-throughput mass spectrometry-based analytical platform for screening large numbers of clones for their chain mispairing levels directly in clarified harvest. Our study of various multispecific constructs revealed that chain mispairing is not only dependent on the sequence and structure, but also a characteristic of the clone and can be mitigated at clone selection stage.

Under current regulatory guidelines, cell-line development for therapeutic proteins is resource and labor intensive.  Integration of high throughput fluorescence-activated cell sorting (FACS), Luminex multi-analyte profiling and Solentim cell imaging methods has enabled analysis of thousands of monoclonal cell-colonies in 96-well plate format.

FACS and imaging avoid time consuming  dilution cloning  experiments and yield a significantly greater assurance of cell monoclonality.  Early clone characterization by highly sensitive Luminex technology allows us to identify the top 20-40 clones from large pools, bypassing intensive intermediate process steps involving several hundred clones.

Following this approach, we completed, in parallel, cell-line development for two antigen variants within time and resource scope of a single antigen, accelerating  the task  by several months compared to traditional cloning timelines. 

The top clones were assessed for scalability and process compatibility in high-throughput automated mini-bioreactors and they met all critical quality attributes. This strategy can be readily implemented as a platform approach for accelerating cell-line development.

Selection of high-productivity clones relies on large-scale screening efforts, examining thousands of cell lines. Despite technological advances, a fundamental understanding of differences between high- and low-productivity clones is still lacking. To address this issue, we evaluated proteomic and epigenetic differences between parental and DHFR-amplified CHO cell lines and among different clonal isolates. We observed global physiological changes in high-productivity clones and differential methylation of the CMV promoter, suggesting avenues for cellular engineering. In particular, increased productivity correlated with substantial changes in ribosomal proteins, endoplasmic reticulum chaperones and primary metabolism.

Product degradation, such as clipping, is a common quality issue in the production of biotherapeutics from Chinese hamster ovary (CHO) cells. In this study, we applied differential quantitative label-free LC-MS/MS proteomic analysis to
understand more fundamentally the intracellular events that may play a role in determining why cell lines from the same cell line development project can vary with regards to the extent of biotherapeutic product clipping.

Recombinant therapeutic proteins are widely used to treat life-threatening diseases including cancer. Chinese hamster ovary (CHO) cells have been used for over 25 years in the biopharmaceutical industry to produce recombinant therapeutics. However, several limitations exist in CHO cell bioprocessing. Here, we discuss using RNA transcriptomics to discover genes important to the protein production process by increasing stabilization. This has potential to decrease costs of medicines by stabilizing CHO genomes.

CHO cell titers have increased over past decades, mainly due to higher cell densities and improved viability. As next-generation processes start to reach the upper limits of cell density, we need to better understand cell-specific productivity (qP) for further gains. Metabolomics tools can help us identify compounds of
interest that are related to qP, which can be used to supplement media or as potential biomarkers to speed clone selection.

Anti-aging protein αKlotho functions as a co-receptor for fibroblast growth factor 23 signaling, whereas its shed extracellular domain (sKlotho), carrying glycosidase activity, is a humoral factor that regulates renal health. Supplementation of sKlotho has emerged as a potential therapeutic strategy for managing kidney disease. This talk will describe how differentiated N-Glycan Modifications on Klotho Protein by CHOand HEK293-modulated protein folding and function, opens up opportunities in therapeutic engineering.

Inducible mammalian expression systems offer an attractive platform for the production of recombinant proteins. The use of an inducible cell line allows the decoupling of growth and the production phases, thereby initially reducing the metabolic load imposed by the expression of the recombinant product. We have conducted process development and intensification for a cumate-inducible GS-CHO cell line and will identify the conditions resulting in enhanced product yield and glycan distribution.