The Centre for Medicines Discovery (CMD), established in August 2020, comprises a number of disease-focused groups and small research facilities (SRF). The biotech facility, provides protein production and mass spectrometry services for academic and industrial customers. In this talk, I will discuss our platforms for production and validation of intracellular, secreted and membrane proteins, highlighting some challenging case studies.

With the ever-increasing demand for antibody and protein-based therapeutics, a flexible purification platform that can handle low to high sample volumes and expression levels is critical for screening. Protein purification using traditional chromatography is limited by throughput and requires labor-intensive sample preparation processes. Magnetic beads-based purification provides a simplified approach to capture target and potentially improving the quality of the purified product. The tools and their application to simplify and augment protein purification and screening cost-effectively will be described.  

Clinical-scale production of biotherapeutics like CAR T and vaccine candidates with high efficacy is crucial for successful therapeutic outcomes. We have developed SUMOstar, a fusion tag technology that enables enhanced expression and secretion of efficacious biotherapeutics in prokaryotic and eukaryotic systems. We demonstrate robust SUMOstar-mediated enhancement of functional anti-human CD19, a CAR T-based cancer therapeutic, in mammalian cell assays. Furthermore, SUMO and SUMOstar can be applied to hyperexpression systems like linear DNA to bypass challenges with conventional plasmids.

Opposing members of the apotosis regulating Bcl-2 protein family critically control the integrity of the mitochondrial outer membrane (MOM) and cellular well-being. To obtain structural insight into the action of the prosurvival Bcl-2 protein and its counterplayer, the pro-death Bax protein, we have developed high-yield expression and purification protocols for those proteins as unlabeled but also uniformly ¹³C/¹⁵N/²H labelled species for structural studies ranging from NMR, X-ray and Cryo-EM to neutron-based methods.

We examined the utility of the recently developed E. coli X-press strain for extracellular protein production. The influence of process parameters on the host’s growth, productivity, leakiness and lysis was studied. E. coli X-press displayed high outer membrane permeability while maintaining viability, constituting a highly promising expression system for extracellular production scenarios. Furthermore, the ecological and economic impact of utilizing E. coli X-press on the early downstream process was analyzed and compared to an intracellular production process. We nicely demonstrate the benefits of extracellular production with E. coli X-press for the downstream process.

Leanbio is committed to the development of biosimilars and new biologic entities, having critical quality attributes and specific productivity of each molecule as key indicators during process development and scale up. Here we present our development strategy following “quality by design” and “yield by design” approaches to rapidly achieve product quality specifications, increase process efficiency and reduce manufacturing process uncertainty for a ‘smooth’ CMC pathway.

The ProteinMaker® system consists of a liquid dispensing station with 24 syringe pumps and a xyz-robotic platform allows the parallel separation of up to 24 proteins. A robot arm allows to pick up eluted samples to purify antibodies using an affinity column followed by a desalting column. A recently developed flow cell allows with LED-technology a simultaneous detection at 280 and 254nm. A description of the system will be presented.

The ALFA system is comprised of a rationally designed epitope tag and three single-domain antibodies. It is suited for a large spectrum of applications in every area of life sciences. NbALFA exhibits an extraordinarily high affinity for the ALFA-tag, making it ideal for imaging and immunoprecipitations. NbALFA^PE and NbALFA^CE with lower affinities allow for the competitive elution of ALFA-tagged proteins at room temperature and 4°C in physiological buffer.

As a Protein Production Core Facility at the Max Planck Institute of Biochemistry, we produce a broad diversity of proteins for many different departments. Particularly challenging are toxic proteins produced at a few µg per liter production scale. We have specifically tailored our workflow to low level expression with respect to affinity tags, expression systems, detection methods and purification procedures. We will present expression and purification of a snake venom L-amino acid oxidase from mammalian cells and the difficulties related to H2O2 generation lethal to the expression host.

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