Drug Discovery – New challenges, new solutions

by in BPS News, BPS Right Sidebar Content

June 2014

MicroorganismsRecombinant and heterologous expression of proteins has been a common practice in industry and academia for the last few decades. It is commonly used with technologies including molecular biology, DNA cloning, protein analytics and purification for discovery of new drugs and drug targets.

Drug Discovery

In drug discovery these input into a range of applications including target validation studies, biochemical assays, structural studies and DMPK measurements. The latter, Drug Metabolism and Pharmacokinetics (DMPK), involves the measurement of a number of pharmacokinetic parameters including bioavailability, drug exposure analysis, and metabolic fate, which describe the fate of compounds in the body. These can be used to compare compounds, to highlight deficiencies in compounds (e.g. high metabolism) and to predict how the potential drug will behave in man – to generate dose predictions. This is particularly of interest to drug developers as the DMPK properties allows them to understand the safety and efficacy data required for the efficient discovery and development of more efficacious , potent and target-directed drugs.

New Process Challenges

The widespread use of these applications in drug development and regulatory approval pose new challenges, for example the need for large amounts of protein for biophysical methods such as NMR (10-500ug), Isothermal Titration Calorimetry (200-5000ug), DSC (100-500ug), and for High Throughput Screening (HTS).  The challenge in the production of large amounts of recombinant protein arises from the complexity of the process. This includes the difficulty in attaining optimal expression in the bacterial expression system with a growing number of recombinant proteins, the impact of using other expression systems further downstream in extracting the protein, the influence of individual species/strain and the effect on downstream cell lysis and purification. Furthermore it is vital to ascertain how much the lysis procedure impacts the expression hosts and whether it forces protein denaturation easily.

The need has also arisen for multi-subunit complexes of proteins for functional activity of a drug target. E.Coli is used as a host for protein expression due to its valued rapid growth rate, simple nutritional requirements and well established genetics; however it is unable to produce a range of recombinant proteins in their soluble form. However on many occasions EColi will produce large multi-domain or dimeric as well as heterologous proteins as insoluble material. This is due to the cytoplasmic environment favouring protein aggregation and proteolysis during or after folding .

Therefore drug discovery has seen an increasing use of eukaryotic expression systems involving insect (Baculovirus, SF9 Spodoptera frugiperda etc) yeast (Saccharomyces cerevisiae, Pichia pastoris etc) as well as mammalian cell lines. The increased use of these expression systems stems from the fact that they are more closely related to animal cells. Therefore they are able to manage recombinant protein synthesis more efficiently than E. coli, resulting in better expression, easier downstream protein extraction and finally achieving stable recombinant proteins.

Cell Disruption Solutions

With a wider range of cell lines with varying cell and protein characteristics and sensitivities, the need for a single solution to offer scalable cell disruption has never been so prominent. The ideal solution should provide control over the environment the cell suspensions are subject to pre- and post- processing to prevent denaturation whilst achieving complete lysis. Moreover it should avoid any successive effects, such as lytic chemicals or detergents biding to proteins, which need further work downstream for attain a functional protein.   The time required for lysis should also be minimal to minimize exposure to proteolytic conditions. In addition there is the ever present need for cost effectiveness, high quality and fast turnaround.

Emulsiflex from Avestin

Avestin C5 High Pressure Homogeniser The Emulsiflex range of high pressure homogenisers from Avestin meets these requirements with cost effective, fast and high throughput systems, with the capabilities to repeatably lyse bacterial, yeast, insect as well as mammalian cell lines. In addition, the range offers a fully controllable “one stop solution” designed with the application and user in mind. Most of all it meets the need to generate high yield as well as high quality intracellular proteins from the hosts, reflected in the use by institutions such as the Structural Genomics Centre (University of Oxford), MRC –Wellcome Trust, Imperial College London as well as AstraZeneca and GlaxoSmithKline.

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