State of the Biomedical Research–Not So Good for Pharma R&D

Exerpt from From Science News

Pfizer’s R&D budget, $9.3 billion in 2010, will drop to less than $8.5 billion this year and to between $6.5 billion and $7 billion in 2012, and the company will stop funding research in internal medicine, allergy and respiratory diseases, urology, and tissue repair.

In fact, the pharmaceutical industry as a whole faces financial pressures, as companies are producing fewer new drugs than in the past. In these conditions, even highly promising research has gotten the ax; in November, Roche cut its RNA interference research unit after spending $400 million over 3 years.

Drug companies also seem less wary nowadays about outsourcing. Among other examples, Eli Lilly began outsourcing animal toxicology studies in 2008, and Wyeth (purchased by Pfizer in 2009) began out sourcing data management for its clinical trials in 2003. In 2007, AstraZeneca even decided to move the production of many active pharmaceutical ingredients—perhaps the core activity of a drug company—to China.

These downsizing events are not particularly caused by still depressed economy, they have more to do with industry-specific patent expiration and productivity issues with large pharmas. What does it all mean to current graduate students and postdocs? Perhaps an even tighter job market than now for starting researchers for some years to come until the next round of sea change comes around. Be aware of what’s going on in smaller, more productive, and focused biotech companies.

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Tags: budget, drug market, patent expiration, Pfizer, R&D, RNAi, Roche

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Making Recombinant Glycosylated Proteins (I)

Many mammalian cell membrane-bound and secreted proteins are glycosylated. The degree of their modifications may be dependent upon tissue specificity and cellular states such as normal vases cancerous. The existence of these proteins in bodily fluids, in combination to their relevance to diseases, makes glycosylated proteins good candidates for clinical diagnostics. Understanding the biogenesis, structure, and functions of these proteins will also aid research and prevention of cancers.

Cancer formation is a heterogeneous and complex process, involving many factors and cellular signaling pathways in each type of cancer. There are more than 1,200 potential cancer biomarkers identified in the literature by a 2006 review. We found that ~ 70% of the 1,261 proteins listed in are naturally secreted proteins, and some 40-50% glycosylated. The addition of carbohydrate groups during protein glycosylation to asparagines (N-linked), or threonines or serines (O-linked) residues may result in mono-, disaccharide- or branched oligosaccharide composed of as many as 20 monosaccharide residues. Glycosylation, together with other modifications, often change the apparent molecular mass of a secreted protein to many folds to that predicted by amino acid sequence. Such heavy modifications on the surface of proteins can influence their functions as well as characteristics as antigens or analytes. Studies of glycosylated proteins offer great opportunities for improving cancer diagnostics.

There are increasing demands for these glycosylated human proteins in good quantity, purity and affordability by the scientific community to perform fundamental and clinical studies in relation to cancer. Such proteins cannot be expressed in bacteria or yeast because those cells do not carry out equivalent post-translation modifications (PTM) as in mammalian cells. Although there have been successful attempts to modify yeast cells to produce proteins with certain types of glycans attached, they were designed for expressing a few pharmaceutical proteins and not suitable for expressing a wide variety of cancer markers. Aside from PTM, expressing human proteins in microorganisms may be hindered by their different codon usage preferences and protein folding tendencies.

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Tags: cancer markers, glycosylated proteins, glycosylation, recombinant proteins, serum samples

New SurfaceBind gDNA Isolation and Purification

Allele Biotech’s SurfaceBind Genomic DNA Pu¬rification Kit is designed for fast, easy, and high-throughput gDNA isolation and purification for lysate obtained through the use of Allele-in-One Mouse Tail Direct Lysis Buffer. Based on our Solid Surface Revers¬ible Binding (SSRB) technology the SurfaceBind system utilizes a plastic tube with its surface coated with proprietary turbo-binders acting to selectively capture and efficiently bind DNA mol¬ecules from reaction mixtures. After lysis of cells, gDNA molecules will specifically interact with the turbo binders and bind to the surface of the tube in the presence of the binding buffer, while pro¬teins and other contaminants will remain in solu¬tion. The DNA can be eluted with as little as 10 microliters of water or buffer for the next application, allowing for a highly concentrated solution.

The entire process of recovery takes less than 10 minutes with only 1 centrifugation step, making it fast and easy. SSRB technology also provides for maxi¬mum DNA capture and release with limited sam¬ple input, without the DNA loss associated with membrane and bead-based technologies.

This is a newly developed product particularly for the Allele Biotech’s customers who use the All-in-One mouse tail genotyping kits: get purified genomic DNA using the same lysate you generated for a quick PCR. The yield and purity will enable direct applications to chip assays, sequencing, Southern blotting, etc.Next time you use Allele-in-One Mouse Tail Direct Lysis Buffer be sure to try our SurfaceBind gDNA Purification kit.

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Finding the Best Capture Reagents

As capture reagents, monoclonal antibodies are the most widely used reagents for specifically detecting and quantifying proteins due to their very high specificity. However, development of monoclonal antibodies is time-consuming and expensive. In addition, many antigens prove to be non-immunogenic or extremely toxic, and therefore cannot be used to generate antibodies in animals. Furthermore, the large size of monoclonal antibodies (150 kDa) may limit their use in cases where more than one binding reagent competes for space to recognize closely juxtaposed epitopes. These limitations could arguably be the biggest hurdles to using monoclonal antibodies as capture reagents for a systematic study of the complete human proteome or for clinical applications of advanced proteomics.

Therefore, alternative capture reagents with high specificity, high affinity, and flexible size and structure that can be easily and cost-effectively produced are urgently needed in order to accelerate proteomic research. Single-chain variable-fragment (scFv) antibodies have been commonly used as alternatives in this regard. scFv is comprised of only the light chain and heavy chain variable regions connected by a peptide linker and with a molecular weight of 27 kDa. Since scFv retains the antigen-binding site of the variable regions, it inherits the specificity of an intact antibody and affinity. In addition, scFv can be easily expressed in yeast or in E. coli with yields in milligrams per liter. scFv can be linked to Fc of desired species specificity and maintain binding properties. If necessary, there is also the option of converting scFv into other antibody formats such as Fab or full IgG by simple cloning steps. The converted antibodies can also be efficiently expressed and purified in yeast or E. coli.

More recently, single domain antibodies that exist in nature were discovered that can be as small as half the size of scFv, and judging from the available data, superior in binding capabilities to scFv or even traditional IgG antibodies. This type of affinity molecules, termed VHH isolated from camelid animals or nurse shark, can be highly expressed in E. coli, linked to a fluorescent protein marker, or chemically conjugated to HRP or other signal generating moieties through a one step reaction.

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