Issue 3 2008

MALDI FT-ICR MS platform for proteomics: Rationale for an offline approach and optimised implementation

Issue 3 2008 / 19 July 2008 /

MALDI FT-ICR MS platform for proteomics: Rationale for an offline approach and optimised implementation
A number of sophisticated approaches have been developed to study the structure and function of genes, including the whole-scale sequencing of entire organisms[1], global transcriptional profiling[2], and forward genetic studies[3]. However, these techniques are ultimately limited by the fact that they only assess intermediates on the way to the protein products of genes that ultimately regulate biological processes[4,5].

Processes such as RNA processing, proteolytic activation, and hundreds of possible post-translational modifications (PTMs) can result in the production of numerous proteins of unique structure and function from a limited number of genes. Additionally, biological activity often results from the assembly of numerous proteins into an active complex, the nature and composition of which can only be explored at the protein level. Therefore, proteomic studies should be able to answer many questions about cellular processes and diseases that cannot be answered by genomic methods alone6.

However, such studies are far harder to perform than their genomic counterparts, and any general analysis platform must possess high sensitivity, be tolerant of a wide range of experimental and analytical conditions, and be able to process and display massive amounts of information. More importantly, these analysis systems must be able to perform extremely high-throughput measurements, since unlike the relatively fixed nature of the genome, the expression and interactions of proteins are in a constant state of flux, varying over time, tissue type, and in response to environmental changes. (more…)

Tagged with: Eric C. Peters, Novartis Institutes for BioMedical Research, Proteomics

High content imaging subpopulation analysis for phenotypic drug discovery

Issue 3 2008 / 19 June 2008 /

Phenotypic drug discovery (PDD) has come of age – again. Using a microscope to observe a cell, one of the oldest techniques available to a cellular biologist dates back to the 17th century studies of Antony van Leeuwenhoek and his characterisation of ‘animalcules’. These early analyses, which simply described the appearance of a cell or group of cells, are the basis for today’s phenotypic assays. Although this early work might seem irrelevant when compared to the powerful array of tools that modern science brings to bear on a problem, the use of cellular phenotype as a method of scientific investigation evolved over time into the primary method of drug discovery up through the 1970s. Cellular phenotypes and the phenotypic changes induced upon compound treatment were commonly followed in both basic science and drug discovery[1-3] and even led to the development of early forms of automated data acquisition and analysis[4].

Although early phenotypic discovery was successful in bringing drugs into the marketplace, in the 1980s the pharmaceutical industry widely switched its focus to a target-based approach in which large chemical libraries were screened against individual enzymatic targets in vitro. This target-based approach to drug discovery (TDD), used in conjunction with high throughput screening and combinatorial chemistry, has remained the industry standard for several decades. Recently, however, productivity in pharmaceutical research and development, as measured by the number of new medical entities (NME), has been declining and it is clear that methods complementary to TDD must be employed5,6. In addition to advances in instrumentation, informatics, and chemical libraries, PDD is now used as an additional method to bring novel therapies to market. PDD is an ideal complement to TDD, where the focus is on a compound’s effect on a single target in vitro, but ignores potential effects on other targets, both positive and negative. (more…)

Tagged with: Eli Lilly, HCS (High Content Screening), Jonathan Low, Louis Stancato, Phenotypic drug discovery (PDD)

Utilisation of secondary screening

Issue 3 2008 / 19 June 2008 /

European Pharmaceutical Review has brought together four individuals from different sides of the scientific palette to discuss current and future issues surrounding secondary screening and maximising its potential. (more…)

Tagged with: Anthony Davies, BioQuant, DiscoveRX Corporation, HCS (High Content Screening), Holger Erfle, Johnson & Johnson, Keith R. Olson, Marc Bickle, Max Planck Institute, Miroslav Cik, Secondary screening, Trinity College Dublin, University of Heidelberg

In vitro toxicity screening as pre-selection tool

Issue 3 2008 / 19 June 2008 /

Drug discovery relies on massive screening of compound libraries with in vitro cell-based target assays. These pharmacological screens have been well accepted. For in vitro toxicological screening, this privilege has only been obtained for the Ames, chromosomal aberration and eye irritation tests. At the moment, a number of cellular assays for cytotoxicity, genotoxicity, embryotoxicity, cellular metabolic activation processes and endocrine disruption await general acceptance. From that point onwards, tools will become available to identify unwanted pharmacological or toxicological effects at a much earlier stage in the drug development process.

One of the challenges of the pharmaceutical industry is to bring more compounds to the market in a shorter space of time. The introduction of combinatorial chemistry and high throughput screening technologies not only resulted in an increased number of compounds, but also a greater diversity of potential drug candidates. Therefore, new, fast and predictive tests are needed to select the best drug candidates.

However, the current success rate of newly developed drugs is too low, as only one out of twenty compounds reaches the market. A superior pre-screening strategy with improved selection criteria would undoubtedly lead to a better choice of drug candidates; thereby proving to be the most appropriate way in which to reduce high costs in clinical development. Therefore, the failures made in the past relating to negative pharmacology and toxicology should be taken into account to improve the future quality of the medicines. Throughout 1990 and 2007, the reasons to stop the development of new drugs changed1. (more…)

Tagged with: Jean Horbach, Schering-Plough Corporation, Toxicology, Walter Westerink, Willem Schoonen

Platform technology for routine three dimensional cell culture

Issue 3 2008 / 19 June 2008 /

Cell culture assays play an important role during the first stages of pharmaceutical development. The design of such in vitro models is significant and data resulting from such tests directly influences the progression of compound development. Therefore it is becoming progressively more important to design cell culture assays that are more representative of the behaviour of cells in living tissues. As a consequence, investigators are developing technology to enhance the cell culture environment and enable cells to grow in ways resembling their in vivo counterparts. This is particularly relevant to the design of culture models that enable three dimensional cell growth in vitro.

The growth of mammalian cells in the tissue culture laboratory provides an important resource for researchers developing new pharmacological reagents. Cell cultures are frequently used as the first step to assess the effect of compounds on living tissues. Our industry is continually looking for new enabling technology that will improve the process of drug discovery, including the development of more accurate in vitro assays. In addition, there is a progressive move toward reducing the numbers of animals used in research and the culture of cells is expected to relieve some of this pressure. The market for cell culture technology is enormous and is expected to rise as a billion dollar industry for years to come1. Accordingly, there is a drive to develop new cell culture technologies, including media formulations, growth supplements and specialised cell culture plastic ware. (more…)

Tagged with: Cell culture automation, Dr Stefan Przyborski, Durham University, ReInnervate Ltd

Peracetic acid in the study of rhIL-11 methionine oxidation

Issue 3 2008 / 19 June 2008 /

Recombinant human interleukin-eleven (rhIL-11) is a pleiotropic cytokine which stimulates stem cell proliferation and affects multiple cell types1. The protein has been demonstrated to provide clinical benefit in platelet restoration2 and Crohn’s disease3. rhIL-11 is a highly basic protein that is rich in arginine, leucine and proline residues, lacks disulfide bonds, is not glycosylated and is not homologous with other known hematopoietic growth factors4. In the development of stable dosage forms of rhIL-11, we have examined the protein’s stability toward methionine oxidation.

Methionine oxidation has been reported as an important instability in several proteins, including human interleukin-5 and human growth hormone5-8. To examine this potential instability, we have utilised reagents that will specifically oxidise methionine residues in an attempt to simulate and predict the oxidation behavior of rhIL-11 on long-term stability. Further, chemical oxidation of rhIL-11 will allow us to understand the ability of various assays to demonstrate stability-indicating properties. The primary sequence of rhIL-11 is provided in Figure 1. The sequence shows two methionine residues (Met58 and Met122). (more…)

Tagged with: Biopharmaceutical, Nicholas W. Warne, Rebecca L. Koval, Thomas J. Crowley, Wyeth Biopharma

Siemens: innovation, industry and implementation

Issue 3 2008 / 19 June 2008 /

Siemens seeks to deliver breakthrough innovations to give customers a unique competitive edge, in turn enabling societies to master their most vital challenges and creating sustainable value. Siemens was one of the first suppliers to adopt the new guidelines of the Food & Drug Administration (FDA) and the European Agency for the Evaluation of Medicinal Products (EMEA), and already possesses in-depth knowledge of the technology needed to achieve desired results within these industry codes and in accordance with the latest GMP requirements. Moreover, Siemens is also an active member of the worldwide pharmaceutical community contributing to the design of new solutions for meeting growing business and production demands. Ingrid Maes from Siemens Headquarters, talks to European Pharmaceutical Review about innovation, industry and implementation. (more…)

Tagged with: Ingrid Maes, Interviews, PAT, Quality by Design (QbD), Siemens

Lyophilization: cycle robustness and process tolerances, transfer and scale up

Issue 3 2008 / 19 June 2008 /

During the past 10-15 years, close attention has been paid to the development of optimal lyophilization cycles for different types of pharmaceuticals[1-4]. Recent advances in process control, such as the Smart Freeze-Dryer^TM technology or similar approaches,[5-7] make cycle development a routine procedure. The attention of many researchers has shifted to the aspects of cycle transfer and scale up that still require significant investment in understanding the differences in lyophilization processes between laboratory and commercial dryers[8-14]. Conducting numerous experiments in an attempt to demonstrate that a laboratory cycle is not only optimal but also robust, requires significant material and time investment. Mathematical modelling of lyophilization processes proved to be a very useful tool, not only for cycle development[15-19] but also for cycle transfer and scale up[11,14]. The same mathematical approach (as discussed in experiment[14]) was applied to the process tolerances design and estimation of cycle robustness in regard to the product temperature. (more…)

Tagged with: Freeze Drying, Lyophilization, Nicholas W. Warne, Serguei Tchessalov, Wyeth Biopharma

Wyeth’s PAT variations submitted through the EMEA Worksharing Procedure

Issue 3 2008 / 19 June 2008 /

In June 2006, the EMEA called for nominations from companies to participate in the pilot phase of a worksharing exercise for Quality variations[1]. Worksharing is a key element in the revisions to the variations proposed by the European Commission, which are intended to streamline the process for making changes to Marketing Authorisations, reducing the regulatory burden and encouraging innovation2. The worksharing procedure is intended where variations to the same nationally authorised product are submitted to the different National Competent Authorities (NCAs). All NCAs are eligible i.e. potentially all 29 EU/EEA states could be involved in the procedure. (more…)

Tagged with: Graham Cook PhD, PAT, Wyeth Pharmaceuticals

Expo sets a new standard in PAT implementation

Issue 3 2008 / 19 June 2008 /

PAT implementation gurus’ Expo Technologies talks to European Pharmaceutical Review about PAT and why they are at the top of their game. (more…)

Tagged with: Dr. Susan Bragg, Expo Technologies, Interviews, PAT, Paul Davies

MicroRNAs for high-throughput functional analysis

Issue 3 2008 / 19 June 2008 /

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules, which are potent post-transcriptional gene expression regulators. They have been shown to participate in the regulation of numerous cellular processes, the list of which is still growing. miRNAs affect numerous targets that can be determined by direct experiments or predicted by bioinformatics approaches, and are presented in several online databases. Feasibility of miRNA for high-throughput experimentation is becoming possible due to the availability of commercially produced molecules, which are able to alter the levels of endogenous miRNAs. miRNA functional analysis will help to validate predicted targets and reveal the role of these small molecules in biological pathways. miRNAs have a high potential to be used as a new gene expression regulating reagent for microscopy based assays.

The discovery of the RNA interference (RNAi) phenomenon a decade ago₁ not only truly revolutionised our understanding about gene regulation processes, but also became a superior tool for unravelling the function of unknown genes_2,3. Around the same time as RNAi, miRNA(s) were discovered₄, and were shown to be potent regulators of gene expression in viruses, animals and plants_5,6. Their regulatory effect is exerted through the pairing to complementing mRNA molecules and inducing translational repression₇. Most miRNAs are thought to make imperfect matching₈, what is largely the reason for their ability to regulate expression of numerous mRNAs simultaneously₉. (more…)

Tagged with: Functional genomics, Holger Erfle, microRNA, University of Heidelberg, Urte Sudžiuviene, Vytaute Starkuviene

PAT – reducing the cost of quality in consumer products manufacturing

Issue 3 2008 / 19 June 2008 /

The ease in making process analytical measurements (typically spectroscopic) in manufacturing has provided a unique opportunity to obtain up-to-date information for making timely process correction decisions. At-line methods provide near-time information without the need for elaborate process control interfacing upfront. This approach works well for batch processing applications or unit operations when process decisions can be made during a defined operation. Effective use of at-line methods includes the measurement of raw materials, premix batches, or quality check point monitoring before finished product is sent to the finishing lines.

However, there is a unique benefit gained in automating spectroscopic measurements and that is the ability to use the information for real-time control of the process which significantly tightens the variability in dosing critical ingredients and reduces the variability in other critical product attributes, such as product moisture.

While the justification for implementing PAT often comes from the ‘realised cost savings’, other benefits of PAT have been identified that are, in many instances, even more important to the bottom line. This area includes the product and process understandings gained that ultimately can improve the effectiveness of an organisation’s quality plan and at lower cost. This paper discusses ways that PAT can reduce the manufacturing costs in the Consumer Products industry as well the benefits found with the implementation of PAT in manufacturing. (more…)

Tagged with: Colgate Palmolive, Debbie Peru, PAT