(185c) Periplasmic Expression of Full-Length Antibodies and Antibody Derivatives in Escherichia Coli Via Multiple Translocation Pathways

Utilizing Escherichia coli (E. coli) for the high-titer production of complex macromolecules such as full-length antibodies (FL-Abs) and antibody derivatives remains a challenge to the biotech industry. Essential to the manufacturing of these therapeutically relevant proteins in E. coli is tight regulation over the expression of the individual light and heavy chains; a process that involves (i) translation by the ribosome as well as (ii) export from the reducing environment of the cytoplasm to the relatively oxidizing milieu of the periplasm. Previous evidence illustrates the ability to gain exceptional control over the translation step of this process through differing promoters as well as manipulation of the translation initiation region (TIR) of the signal peptide via the generation of degenerate, wobble-based codon libraries. In this work, we develop novel TIR signal peptide libraries through both empirical means as well as rational design based on predicted mRNA secondary structures and their associated free-energies. We demonstrate that optimization of antibody chain expression by TIR management coupled with N-terminal fusions of these chains to signal peptides known to use various methods for translocation across the inner-membrane (i.e. co- and post-translational) greatly alters functional expression of FL-Abs and their derivatives in E. coli. Additionally, we use reversed-phase high pressure liquid chromatography (RP-HPLC) to quantify intracellular soluble and insoluble antibody chain titers in order to gain a deeper understanding of how the signal peptide and/or the TIR effects the partitioning of an over-expressed, heterologous protein between these two fractions. This work provides new insights into the challenges associated with non-native protein expression in E. coli at both the manufacturing and lab bench scales.