Strategies to boost cellular antioxidant capacity appear to be beneficial for recombinant protein expression. Here we showcase the use of a novel hydrogen-peroxide evolved CHO host in improving the expression of two difficult-to-express bispecific antibodies. We demonstrate that these improvements are linked to enhanced antioxidant capacity through the upregulation of diverse antioxidant defense genes as well as improved glutathione content.

Lipid metabolism plays a key role in cellular processes, such as endoplasmic reticulum size and secretion, which are central to achieving high recombinant protein titres. We have developed a selection system based on the requirement of CHO cells to produce proline and employed this system to genetically engineer lipid metabolism in CHOK1-SV cells which resulted in endoplasmic reticulum expansion and enhanced recombinant protein titres between 1.5 and 9-fold.

Recombinant CHO cell line development (CLD) has been a crucial step for therapeutic protein production platforms; however, this step remains time-consuming and costly. With the emergence of genome editing technology such as CRISPR/Cas9, site-specific integration has been successfully implemented in CHO cells for expediting the process of CLD. This presentation describes the trends in CHO CLD from random to targeted approaches. And I will cover the major obstacles faced in rational CHO CLD and the potential strategies employed to overcome its limitations. Finally, I conclude by discussing future directions and challenges for next-generation CHO cell factories.

• NEW DATA - This Presentation Contains New Data
  

High producing stable mammalian cells are often required for producing recombinant proteins. Traditional methods like antibiotic selection cause high levels of cell stress during the selection or require high initial costs for instruments. Here, we present a new bead based method for generating high producing stable cell pools that avoids substantial negative effects on viability or cell growth after selection and that does not have high acquisition costs for instruments. Selected pools showed a threefold higher expression than puromycin selected cells. However, this method can also be combined with other selection technologies. *NEW DATA – This Presentation Contains New Data

Recombinant expression requires cells in balance for quality and yield. We present a toolbox for balancing protein translation in mammalian cells by RNA regulation elements (RgE) in the 5'-UTR. RgEs varying in thermodynamic stability lead to predictable dosage from 6-110% in mammalian cell lines. Applying these to tune expression of IgG heavy/light chains lead to up to 3.5-fold increased titers and reduction of IgG aggregates.

During the live cycle of a biopharmaceutical project the production needs to stay up to date with productivity and quality demands from early pre-clinical to market phase. Optimization can be done at different stages and on different levels with selecting the right cell line, selecting the right clone or optimizing the media/feed combination and / or optimizing process parameters. We provide case studies which address the different possibilities of optimization.

CHO cell line selection represents a painful bottleneck in biotherapeutic development, particularly for complex molecules like bispecifics. The Opto™ CLD workflow on the Beacon® system accelerates early CLD by integrating high throughput cell sorting, cloning, culture, productivity, growth, and product quality assays into a single, 5-day automated process. We will introduce new capabilities including on-chip detection of product aggregates and contaminating byproducts that pinpoint the best clones early on – saving valuable development time while de-risking the path to IND.

Low temperature expression is an effective strategy to improve the solubility of aggregation-prone "difficult-to-express" proteins especially in case of the expression using E. coli. Glutathione S-transferase (GST) has been widely adopted as an affinity fusion tag, which has also been shown to promote the solubility of its fusion partners. We have developed engineered GSTs that can dramatically enhance the protein expression at low temperature. Our engineered GST is highly suitable for expressing and purifying proteins that tend to aggregate and/or be insoluble at normal temperature (30-37 ºC) if it combined with the low temperature expression strategy (10-25 ºC).

• NEW DATA - This Presentation Contains New Data
  

Lactococcus lactis bacteria have been engineered such that they can produce lanthionine-containing GPCR agonists. The lanthionine-constraint confers high GPCR specificity and in vivo stability. LP2 a lanthipeptide agonist of the angiotensin II type 2 receptor had in man a T1/2 of >2 hours in contrast to seconds of the natural ligands. High therapeutic efficacy in animal models of multiple diseases has been demonstrated for lanthionine-constrained peptide agonists of GPCRs.

Technology platforms are integral to the design, development and commercialization of recombinant biotherapeutics. We developed a versatile platform - SUREtechnology – that facilitates the effective combination of polypeptides. It provides convenience to produce complex protein scaffolds including antibodies, bispecifics, viruses, Fc-fusions, cytokines, and vaccine antigens. By enabling a robust expression of transgenes and enhanced throughput it enables the combination with metabolism interfering proteins to ease the expression of difficult-to-express scaffolds.

Our unique platform technology named PUREfrex is a fully reconstituted (or rebuilt) cell-free protein expression system. It's easy to customize the system for various applications, and useful for high throughput screening of various kinds of biologics as well. Plus, we established robust ribosome display with customized PUREfrex named PUREfrexRD, which has great advantage in screening of highly diversified library to generate new antibodies or cyclic peptides.