# Application of the iPLUS non-coding sequence in improving biopharmaceuticals production

## TL;DR

This paper shows that a non-coding sequence called iPLUS can significantly boost the production of biopharmaceuticals like monoclonal antibodies and mRNA vaccines.

## Contribution

The study introduces iPLUS as a universal cis-regulatory element that enhances recombinant protein and mRNA production across multiple biopharmaceutical systems.

## Key findings

- Incorporating iPLUS in a monoclonal antibody vector increased trastuzumab production by 2-fold in ExpiCHO cells.
- Using iPLUS in Pichia pastoris increased reporter protein production by over 100-fold.
- iPLUSv2 doubled Spike and MAGEC2 protein production in mRNA vaccine contexts.

## Abstract

The biotechnological landscape has witnessed significant growth in biological therapeutics particularly in the field of recombinant protein production. Here we investigate the function of 3′UTR cis-regulatory elements in increasing mRNA and protein levels in different biological therapeutics and model systems, spanning from monoclonal antibodies to mRNA vaccines. We explore the regulatory function of iPLUS - a universal sequence capable of consistently augmenting recombinant protein levels. By incorporating iPLUS in a vector to express a monoclonal antibody used in immunotherapy, in a mammalian cell line used by the industry (ExpiCHO), trastuzumab production increases by 2-fold. As yeast Pichia pastoris is widely used in the manufacture of industrial enzymes and pharmaceuticals, we then used iPLUS in tandem (3x) and iPLUSv2 (a variant of iPLUS) to provide proof-of-concept data that it increases the production of a reporter protein more than 100-fold. As iPLUS functions by also increasing mRNA levels, we hypothesize that these sequences could be used as an asset in the mRNA vaccine industry. In fact, by including iPLUSv2 downstream of Spike we were able to double its production. Moreover, the same effect was observed when we introduced iPLUSv2 downstream of MAGEC2, a tumor-specific antigen tested for cancer mRNA vaccines. Taken together, our study provides data (TLR4) showing that iPLUS may be used as a valuable asset in a variety of systems used by the biotech and biopharmaceutical industry. Our results underscore the critical role of non-coding sequences in controlling gene expression, offering a promising avenue to accelerate, enhance, and cost-effectively optimize biopharmaceutical production processes.