Advances in Cell-Free Protein Synthesis (CFPS) offer the prospect of industrially relevant screening and even production processes, with the advantage of greater speed, control over process environment, integration into high-throughput systems and improved consistency. We present studies of how biochemical engineering approaches can be used to improve the titre and quality of a virus-like particle (VLP) protein product, by manipulation of process environment parameters and plasmid design.

Although monoclonal antibodies are considered as standard molecules in biotherapeutic industry, some of those drugs are still difficult-to-express molecules. Here, we are highlighting the development of a difficult-to-express monoclonal antibody and its challenges for CHO cell line generation. Therefore, protein engineering and modulation of vector architecture are presented as approaches to improve product yields. Moreover, a new cell line development concept in CHO going beyond random transgene integration is highlighted to further boost the expression of a difficult-to-express molecule.

E. coli, the work horse host for protein production still faces many challenges in protein expression.  Therefore, we have engineered strains for the standardized 2-stage expression of heterologous proteins (in stationary phase). 2-stage production enables the utilization of 2-stage dynamic control, wherein the cellular state can be modified  (beyond conditions compatible with growth) to enable optimal protein expression, even with difficult to express proteins. Level of key proteases, chaperones and even the reduction potential of the cytoplasm can be dynamically controlled.  We demonstrate the broad applicability of this approach to express a variety of protein targets of commercial interest.

In the new era of complex modalities and COVID-19 pandemic, speed, capacity, efficiency, and robustness are more vital than ever to a successful biotherapeutics development program. Yet CHO cell line selection still represents a painful, costly bottleneck - requiring weeks waiting for cells to expand and processing of hundreds of well plates. Learn how Opto™ CLD workflow on the Beacon® system integrates cell enrichment, cloning, culture, screening, and selection into a single, automated microscale process that generates the highest titer clones in just a few days. Case studies will highlight how users are accelerating and de-risking their path to IND with capacity for up to 50 campaigns per year, multiple molecules per campaign, and FDA-accepted monoclonality assurance to support regulatory submissions.

The intrinsic pathway of programmed cell death is tightly regulated by the Bcl-2 protein family located at the mitochondrial outer membrane, controlling the release of apoptotic factors. For a long time, obtaining enough Bcl-2 membrane protein for biophysical studies was difficult, which demanded a more efficient purification protocol. This was finally solved by using a classical Escherichia coli expression system, increasing the yield to produce 10-20 mg of full-length protein.

Pelican Expression Technology (formerly Pfenex) is a validated, cost-effective, and scalable platform for recombinant protein production and is well-suited for large-scale protein production, where traditional systems are not suitable. Learn how this Pseudomonas-based expression platform was developed for recombinant protein production. Case studies presented demonstrate the genetic elements, host strains, and automated strain screening workflows enabled broad exploration of expression strategies for two complex antibody scaffolds: Fab fragment and multimeric nanobodies.

Membrane proteins are important drug targets (GPCRs, ion channels), yet are notoriously difficult to work with. We have developed a novel one-step approach for the incorporation of membrane proteins directly from crude cell membranes into lipid Salipro particles. This direct approach termed Salipro DirectMX presents new opportunities for de novo development and characterisation of biologics and small molecules, including phage display, B-cell sorting and cryoEM.

Affinity tags have been widely applied to purification of G protein-coupled receptors (GPCR) for structural studies. We developed a novel calcium-dependent EF-based affinity system that allows capture and high recovery of GPCR from dilute solutions l. The binding of the EF1 tag to the resin is very strong (high picomolar to low nanomolar range) that allows efficient purification without any loss of the target protein.

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.

CD19 on malignant B cells constitutes the target of approved CAR T cell-based cancer immunotherapies, but assessment of CAR T cells is hampered by limited availability of CD19. We expressed a novel fusion construct consisting of the full-length extracellular domain of CD19 and domain two of human serum-albumin (CD19-AD2) in CHO and optimized production by continuous fermentation. Purified monomeric CD19-AD2 showed specificity to CAR T cells and also activation of T cell function.

Often the first activity within a new project is the generation of protein reagents for assay development, antibody discovery or structural studies.  Giving meaningful project timelines for such activities is challenging as it is almost impossible to predict how long it will take to express and purify a protein.  Using worked examples, I will highlight the unpredictability of recombinant protein production and share details on the processes implemented at UCB to increase chances of success.

We have developed a unique recombinant protein secretion system by using Corynebacterium glutamicum and protein expression service Corynex^®. It has some advantages such as the secretion of correctly folded and active form, high purity, and endotoxin-free. Recently, “Antibody mimetics” have been developing as well as conventional major antibodies, so we have examined the production of those proteins. We demonstrate Corynex^® is very suitable system to produce various kinds of antibody mimetics.

Protein purification has been made much easier by the incorporation of many different tags, but most tags don’t have other functions beyond facilitating the purification. Here we show the modifications of a protein, recombinant human papilloma virus 16 L1 (rHPV 16 L1), that not only improve the purification of the modified protein but also afford the protein with desired immunological properties.