A dynamic mathematical model for monoclonal antibody N-linked glycosylation and nucleotide sugar donor transport within a maturing Golgi apparatus.

Monoclonal antibodies (mAbs) are one of the most important products of the biopharmaceutical industry. Their therapeutic efficacy depends on the post-translational process of glycosylation, which is influenced by manufacturing process conditions. Herein, we present a dynamic mathematical model for mAb glycosylation that considers cisternal maturation by approximating the Golgi apparatus to a plug flow reactor and by including recycling of Golgi-resident proteins (glycosylation enzymes and transport proteins [TPs]). The glycosylation reaction rate expressions were derived based on the reported kinetic mechanisms for each enzyme, and transport of nucleotide sugar donors [NSDs] from the cytosol to the Golgi lumen was modeled to serve as a link between glycosylation and cellular metabolism. Optimization-based methodologies were developed for estimating unknown enzyme and TP concentration profile parameters. The resulting model is capable of reproducing glycosylation profiles of commercial mAbs. It can further reproduce the effect gene silencing of the FucT glycosylation enzyme and cytosolic NSD depletion have on the mAb oligosaccharide profile. All novel elements of our model are based on biological evidence and generate more accurate results than previous reports. We therefore believe that the improvements contribute to a more detailed representation of the N-linked glycosylation process. The overall results show the potential of our model toward evaluating cell engineering strategies that yield desired glycosylation profiles. Additionally, when coupled to cellular metabolism, this model could be used to assess the effect of process conditions on glycosylation and aid in the design, control, and optimization of biopharmaceutical manufacturing processes.

Development of reagents and assays for the detection of pathogenic Burkholderia species.

Rapid detection of the category B biothreat agents Burkholderia pseudomallei and Burkholderia mallei in acute infections is critical to ensure that appropriate treatment is administered quickly to reduce an otherwise high probability of mortality (ca. 40% for B. pseudomallei). We are developing assays that can be used in clinical laboratories or security applications for the direct detection of surface-localized and secreted macromolecules produced by these organisms. We present our current medium-throughout approach for target selection and production of Burkholderia macromolecules and describe the generation of a Fab molecule targeted to the B. mallei BimA protein. We also present development of prototype assays for detecting Burkholderia species using anti-lipopolysaccharide antibodies.

CE-based sample quality assessment prior to 2-D gel electrophoresis: towards the standardization of gel-based proteomics.

2-DE remains one of the most commonly used separation techniques for complex protein mixtures. This article describes a new approach to 2-DE sample assessment using SDS capillary gel electrophoresis (in Beckman Coulter sieving medium) combined with multi-pixel detection. The performance of this platform was investigated using protein samples prepared for 2-DE. The capability to characterize 2-DE sample was tested and the results show that the repeatability of peak migration time and intensity are better than 2% RSD. The system gives good resolution, accurate molecular mass assignment, as well as absolute and relative quantification of proteins. Notably, this study also demonstrates the use of this platform to screen the quality of simple and complex 2-DE samples. Implementation of this technique in the proteomics workflow will not only improve the success rate of 2-DE, but will also enable sample verification before 2-DE and allow the relative quantification of proteins. The validation of differential protein expression is also demonstrated using the combined information of relative molecular mass and relative quantification. It is the first time that a rapid and visual evaluation method is reported for the quality assessment of 2-DE samples.

Towards the implementation of quality by design to the production of therapeutic monoclonal antibodies with desired glycosylation patterns.

Quality by design (QbD) is a scheme for the development, manufacture, and approval of pharmaceutical products. The end goal of QbD is to ensure product quality by building it into the manufacturing process. The main regulatory bodies are encouraging its implementation to the manufacture of all new pharmaceuticals including biological products. Monoclonal antibodies (mAbs) are currently the leading products of the biopharmaceutical industry. It has been widely reported that glycosylation directly influences the therapeutic mechanisms by which mAbs function in vivo. In addition, glycosylation has been identified as one of the main sources of monoclonal antibody heterogeneity, and thus, a critical parameter to follow during mAb manufacture. This article reviews the research on glycosylation of mAbs over the past 2 decades under the QbD scope. The categories presented under this scope are: (a) definition of the desired clinical effects of mAbs, (b) definition of the glycosylation-associated critical quality attributes (glycCQAs) of mAbs, (c) assessment of process parameters that pose a risk for mAb glycCQAs, and (d) methods for accurately quantifying glycCQAs of mAbs. The information available in all four areas leads us to conclude that implementation of QbD to the manufacture of mAbs with specific glycosylation patterns will be a reality in the near future. We also foresee that the implementation of QbD will lead to the development of more robust and efficient manufacturing processes and to a new generation of mAbs with increased clinical efficacy.

HUPO Biological Reference Material Initiative Workshop 26 September 2009, Toronto, Canada.

The Biological Reference Material Initiative Workshop held at the Toronto HUPO congress on 26 September 2009, focused on the development of new biological reference materials and tools for the assessment of reproducibility, the solutions to many of the technical challenges in proteomics and protein-based molecular diagnostics. This half-day meeting included presentations from leading scientists from the worldwide proteomic community, who shared a common interest in standardization and increased accuracy of proteomic data. The conclusion was that proteomics is highly sensitive to both biological and technical variability. It is this biological and technical variance, when not accounted for by experiment design, that invalidates proteomic experiments, but both of these issues can be dealt with by tackling reproducibility.

Comparison of three commercially available DIGE analysis software packages: minimal user intervention in gel-based proteomics.

The success of high-performance differential gel electrophoresis using fluorescent dyes (DIGE) depends on the quality of the digital image captured after electrophoresis, the DIGE enabled image analysis software tool chosen for highlighting the differences, and the statistical analysis. This study compares three commonly available DIGE enabled software packages for the first time: DeCyder V6.5 (GE-Healthcare), Progenesis SameSpots V3.0 (Nonlinear Dynamics), and Dymension 3 (Syngene). DIGE gel images of cell culture media samples conditioned by HepG2 and END2 cell lines were used to evaluate the software packages both quantitatively and subjectively considering ease of use with minimal user intervention. Consistency of spot matching across the three software packages was compared, focusing on the top fifty spots ranked statistically by each package. In summary, Progenesis SameSpots outperformed the other two software packages in matching accuracy, possibly being benefited by its new approach: that is, identical spot outline across all the gels. Interestingly, the statistical analysis of the software packages was not consistent on account of differences in workflow, algorithms, and default settings. Results obtained for protein fold changes were substantially different in each package, which indicates that in spite of using internal standards, quantification is software dependent. A future research goal must be to reduce or eliminate user controlled settings, either by automatic sample-to-sample optimization by intelligent software, or by alternative parameter-free segmentation methods.

Proteomic characterization of the conditioned media produced by the visceral endoderm-like cell lines HepG2 and END2: toward a defined medium for the osteogenic/chondrogenic differentiation of embryonic stem cells.

Visceral endoderm (VE) is an extraembryonic cell layer formed before gastrulation that secretes critical factors involved in embryonic development with inductive effects on embryonic stem cell (ESC) differentiation. We utilized the conditioned media (CM) from the VE-like cell lines, HepG2 and END2, to enhance lineage-specific differentiation of murine ESCs (mESCs) toward the osteogenic lineage. Previously, we have demonstrated that use of the HepG2-CM resulted in the efficient osteogenic/chondrogenic differentiation of mESCs without embryoid body (EB) formation. In this study, we demonstrate, for the first time, the osteogenic-inducing activity of END2-CM, suggesting a potential shared protein profile between HepG2- and END2-CM. To identify the active factors in the CM, proteomic analysis using differential gel electrophoresis coupled with matrix-assisted laser desorption/ionization was performed on the two CM, resulting in six proteins being established to be present in both CM, including ones that may function on the epithelial-mesenchymal transition (EMT). Our results represent the first study on the VE-like cell line secretome and provide information on VE proteins identifying possible candidate proteins to be used for mesoderm-specific osteogenic differentiation.

2nd BSPR London Regional Meeting and 6th Annual Imperial Proteomics Day. November 2007, Imperial College London, London, UK.

The 2(nd) BSPR London Regional Meeting held at Imperial College London focused on nanoproteomics and single cell proteomics, the solutions to many of the technical challenges in proteomics and protein based molecular diagnostics. This one day meeting included presentations from leading scientists within and outside of Imperial College who share a common interest in novel solutions for the identification and characterization of proteins from a single cell. The conclusion was that nanomaterials are delivering enhanced reagents and have been tested at the proof-of-concept level, but have yet to be incorporated into routine proteomic workflows.

Proteomics, nanotechnology and molecular diagnostics.

Sequencing of the human genome opened the way to the exploration of the proteome and this has lead to the identification of large numbers of proteins in complex biological samples. The identification of diagnostic patterns in samples taken from patients to aid diagnosis is in the early stages of development. The solution to many of the technical challenges in proteomics and protein based molecular diagnostics will be found in new applications of nanomaterials. This review describes some of the physical and chemical principles underlying nanomaterials and devices and outlines how they can be used in proteomics; developments which are establishing nanoproteomics as a new field. Nanoproteomics will provide the platform for the discovery of next generation biomarkers. The field of molecular diagnostics will then come of age.

Towards microfluidic technology-based MALDI-MS platforms for drug discovery: a review.

BACKGROUND: Microfluidic methods have found applications in various disciplines. It has been predicted that the microfluidic technology would be useful in performing routine steps in drug discovery ranging from target identification to lead optimisation in which the number of compounds evaluated in this regard determines the success of combinatorial screening. The sheer size of the parameter space that can be explored often poses an enormous challenge. OBJECTIVE: We set out to find how close we are towards the use of integrated matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) microfluidic systems for drug discovery. METHODS: In this article we review the latest applications of microfluidic technology in the area of MALDI-MS and drug discovery. RESULTS/CONCLUSIONS: Our literature survey revealed microfluidic technologies-based approaches for various stages of drug discovery; however, they are in still in developmental stages. Furthermore, we speculate on how these technologies could be used in the future.

Highlights of the first BSPR London Regional Meeting and Fifth Imperial Proteomics Day.

Now that the genomics revolution is tailing off proteomics promises an even more radical transformation of biological and medical research. Ultimately, the clues to the mysteries of biological processes will lye within the proteins present at a given time. Proteomics, the characterization of complex protein mixtures, builds on a wide range of expertise from protein chemistry to mass spectrometry and bioinformatics. Each step from sample preparation, high resolution protein separation to protein identification is underpinned by the latest technological developments in protein separation and mass spectrometry and supported by powerful computational image and data analysis algorithms which requires large scale data management and storage facilities. The success of proteomics depends on further developments in technology which need a wide range of scientific expertise. This meeting, the first BSPR London Regional Meeting, brought together scientists from a broad background who share a common interest in novel solutions for the characterization of complex protein mixtures.

Paraoxonase gene polymorphism and serum activity in progressive IgA nephropathy.

BACKGROUND: HDL-associated paraoxonase (PON1) reduces oxidation of lipids in LDL, and activity is inversely related to coronary heart disease risk with a beneficial effect on the development of atherosclerosis. Risk factors associated with atherosclerosis, such as hypertension, dyslipidemia and smoking, also promote the progression of chronic glomerulonephritides which may therefore be associated with perturbations in PON1 activity. METHODS: We performed a genetic association study in patients with IgA nephropathy (IgAN) (n=115) compared with control subjects (n=118). The aim was to test whether polymorphisms in the PON1 coding region (Q192R and L55M) and its promoter (-108C/T and -162A/G) are associated with either IgAN or with the progression. We measured serum paraoxonase activity in 60 out of 115 patients. All patients had been followed up for more than 4 years. RESULTS: There were no differences in the genotype frequency at 3 of the polymorphic sites (Q192R, L55M and -108C/T) between the patients and controls. However, the frequency distribution at -162 position (A/G) was significantly diffe-rent in IgAN (p=0.028, chi-square test) with a higher frequency of the heterozygote (0.017, Fisher exact test [FE]; odds ratio [OR] = 1.99; 95% confidence interval [95% CI], 1.14-3.47). Although there were no differences in the genotype frequency at 3 of the polymorphic sites (Q192R, L55M and -162C/T) between the patients with progressive IgA and the nonprogressive patients, we found that the frequency of the C allele for the -108C/T polymorphism was elevated in those patients with nonprogressive disease (n=85) compared with those with progressive disease (n=30) (61% vs. 47%; p=0.070, FE; OR=1.75, 95% CI, 0.97-3.18). Furthermore, PON1 activity was significantly higher in nonprogressive patients compared with progressors (206 +/- 71 vs. 136 +/- 48; p<0.001), and activity significantly correlated with 1/serum creatinine (SCr) (p<0.001; r=0.38). CONCLUSIONS: The results of this study suggest that in IgAN, lower PON1 activity may be associated with the deterioration of kidney function. This could be due to variable expression of the PON1 gene, or a functional effect of the gene product.

Probing the function of Mycobacterium tuberculosis catalase-peroxidase by site-directed mutagenesis.

Catalase-peroxidase is a multi-functional heme-dependent enzyme which is well known for its ability to carry out both catalatic and peroxidatic reactions. Catalase-peroxidase from Mycobacterium tuberculosis(mtCP) is of particular interest because this enzyme activates the pro-antitubercular drug isoniazid. It is estimated that 2 billion people are infected with M. tuberculosis, the principal causative agent of tuberculosis, and that 2 million people die from the disease each year. The rise of drug-resistant strains continues to be of critical concern and it is well documented that mutations which reduce activity or inactivate mtCP lead to increased levels of isoniazid resistance in M. tuberculosis. The recent determination of the crystal structure for M. tuberculosis mtCP has aided the understanding of how the enzyme functions and provides a three-dimensional framework for testing hypotheses about the roles of various residues in the active site. Here we report site-directed mutagenesis studies of three conserved residues located near the heme of mtCP, His-108, Trp-107 and Trp-321 including the construction of the double mutant W107F-W321F. Resulting mutants have been purified and their catalatic and peroxidatic activities have been determined. Data are compared in the context of related studies aimed at dissecting the roles of these residues in the different activities of the enzyme. Analyses of single and double mutants studied here emphasise that the hydrogen bonding network surrounding the heme in the active site appears more important for maintenance of catalatic rather than peroxidatic activity in CP enzymes.

Crystal structure of Mycobacterium tuberculosis catalase-peroxidase.

The Mycobacterium tuberculosis catalase-peroxidase is a multifunctional heme-dependent enzyme that activates the core anti-tuberculosis drug isoniazid. Numerous studies have been undertaken to elucidate the enzyme-dependent mechanism of isoniazid activation, and it is well documented that mutations that reduce activity or inactivate the catalase-peroxidase lead to increased levels of isoniazid resistance in M. tuberculosis. Interpretation of the catalytic activities and the effects of mutations upon the action of the enzyme to date have been limited due to the lack of a three-dimensional structure for this enzyme. In order to provide a more accurate model of the three-dimensional structure of the M. tuberculosis catalase-peroxidase, we have crystallized the enzyme and now report its crystal structure refined to 2.4-A resolution. The structure reveals new information about dimer assembly and provides information about the location of residues that may play a role in catalysis including candidates for protein-based radical formation. Modeling and computational studies suggest that the binding site for isoniazid is located near the delta-meso heme edge rather than in a surface loop structure as currently proposed. The availability of a crystal structure for the M. tuberculosis catalase-peroxidase also permits structural and functional effects of mutations implicated in causing elevated levels of isoniazid resistance in clinical isolates to be interpreted with improved confidence.

Proteomics: New Developments in Target Discovery.

The Society for Medicines Research in collaboration with the Biological and Medicinal Chemistry sector of the Royal Society for Chemistry held a meeting on September 19, 2002, in London, United Kingdom to discuss proteomics in drug discovery. The meeting gave the most up-to-date overview of current progress in this new field, the challenges in silico, in vitro and in vivo, together with consideration of the increasing contribution of bioanalysis, bioinformatics and pharmacogenomics. Speakers from Celera Genomics, Oxford GlycoSciences and GlaxoSmithKline, among other companies and institutions, were present. (c) 2002 Prous Science. All rights reserved.