In November 2021, Rathangadhara Nammalwar (manager of protein-based therapeutics at Sartorius) delivered a presentation describing the hallmarks of a robust cell line development (CLD) platform. Focusing on the importance of expression constructs, he then explained how collaboration with external partners has enabled Sartorius to optimize its Cellca CLD platform for mammalian-cell production of therapeutic proteins.
Nammalwar identified key criteria for selecting a CLD platform, noting first that it should leverage a highly productive cell line that yields high-quality proteins. All raw materials should have fully traceable lineages and come from antibiotic-free processes. The period from gene synthesis to production of a research cell bank (RCB) should be reasonably short. Ensuring platform scalability can bolster productivity and help to reduce time, labor, and costs for media and process optimization.
To meet such criteria, Sartorius has enhanced the Cellca CLD platform. It features a dhfr-deficient Chinese hamster ovary (CHO) DG44 cell line that exhibits long-term stability. The cells have been adapted to suspension culture in chemically defined media. Supporting those technologies are dedicated media-development and process-design teams who work with clients to define culture requirements and ensure process scalability upon technology transfer.
Improvements to the platformâ€™s expression vectors have enhanced CLD outcomes, as has application of distinct vectors for single- and multichain proteins. A third-generation vector had featured systems for metabolic selection and for stringent selection of dihydrofolate reductase (DHFR), optimized scaffoldâ€“matrix attachment regions (S/MARs), and antibiotic-free processes. Sartorius recently collaborated with UniTargetingResearch (a Norwegian vector-design company) to optimize the constructâ€™s promoters, 5â€˛ and 3â€˛ untranslated regions (UTRs), and signal peptides (SPs). Nammalwar explained that UTRs influence mRNA stability and translation efficiency. SPs guide translocation of newly synthesized proteins. Nammalwar explained that modifying such elements has increased the vectorâ€™s productivity significantly. Equally important is that Sartorius and UniTargetingResearch determined the need to apply different UTR sequences for single- and multichain proteins.
Development of the fourth-generation vectors began with cloning different UTRTech and UTRBetatech sequences (UniTargetingResearch) into Sartoriusâ€™s existing construct, with plasmids encoding for a model Fc fusion protein or IgG1 antibody. The vectors were transfected with CHO cells to generate large pools, cells from which were expanded in shake flasks. High-performing batches were selected for fed-batch cultivation in shake flasks, after which titers were measured.
Vectors with UTRBetatech EC 20 yielded cells producing 40% more fusion protein than did the standard construct. Vectors containing UTRBetatech EC 11 generated cells that expressed 50% more IgG1 than did the standard. Similar results came from testing other proteins and antibodies.
The top-performing vectors underwent further large-pool studies to assess the influences of SPs. For three fusion proteins, Sartorius tested batches using its standard plasmid, a vector bearing UTRBetatech EC 20, and a construct containing that and a sequence for a customer-specified SP. Batches made using the UTRBetatech vector alone showed 1.5â€“2Ă— higher titers than those made using the standard. Similar studies were conducted for three antibodies but using Cellca SP(9) rather than a customer-specified peptide. Vectors combining the UTRBetatech EC 11 and Cellca SP(9) sequences outperformed other tested options, resulting in titers 2â€“3Ă— greater than those achieved using a third-generation construct.
Having observed promising results from studies of clone performance, Sartorius has introduced its latest constructs into an optimized workflow comprising vector construction, nucleofection, minipool generation, single-cell cloning by fluorescence-activated cell sorting (FACS), clone screening based on cultures in Ambr 15 bioreactors, and RCB generation. Often, the process can be completed in 14 weeks, and resulting cell lines have been highly productive and scalable.
Questions and Answers
Why does Sartorius use distinct UTRs for single- and multichain proteins? Early development studies indicated that using the same UTR limited expression of single-chain proteins. Thus, we also sought out UTR sequences specific to such products.
Why should a vector include an optimized UTR and SP? Our development studies show that incorporating both elements is important. Optimizing a UTR alone can increase process productivity but might not generate the highest possible titers.
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