Essentially Leading Antibody Production: An Investigation of Amino Acids, Myeloma, and Natural V-Region Signal Peptides in Producing Pertuzumab and Trastuzumab Variants.

Boosting the production of recombinant therapeutic antibodies is crucial in both academic and industry settings. In this work, we investigated the usage of varying signal peptides by antibody V-genes and their roles in recombinant transient production, systematically comparing myeloma and the native signal peptides of both heavy and light chains in 168 antibody permutation variants. We found that amino acids count and types (essential or non-essential) were important factors in a logistic regression equation model for predicting transient co-transfection protein production rates. Deeper analysis revealed that the culture media were often incomplete and that the supplementation of essential amino acids can improve the recombinant protein yield. While these findings are derived from transient HEK293 expression, they also provide insights to the usage of the large repertoire of antibody signal peptides, where by varying the number of specific amino acids in the signal peptides attached to the variable regions, bottlenecks in amino acid availability can be mitigated.

The quantification of antibody elements and receptors subunit expression using qPCR: The design of VH, VL, CH, CL, FcR subunits primers for a more holistic view of the immune system.

The expression levels of immunoglobulin elements and their receptors are important markers for health and disease. Within the immunoglobulin locus, the constant regions and the variable region families are associated with certain pathologies, yet a holistic view of the interaction between the expressions of the multiple genes remain to be fully characterized. There is thus an important need to quantify antibody elements, their receptors and the receptor subunits in blood (PBMC cDNA) for both screening and detailed studies of such associations. Leveraging on qPCR, we designed primers for all Vκ1-6, VH1-7, Vλ1-11, nine CH isotypes, Cκ, Cκ, Cλ1 &3, FcεRI α,ß, and γ subunits, all three FcγR and their subunits, and FcαR. Validating this on a volunteer PBMC cDNA, we report a qPCR primer set repertoire that can quantify the relative expression of all the above genes to the GAPDH housekeeping gene, with implications and uses in both clinical monitoring and research.