Judit M. Nagy

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.

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.

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.

Purification and characterization of recombinant catalase-peroxidase, which confers isoniazid sensitivity in Mycobacterium tuberculosis.

The Mycobacterium tuberculosis katGgene encodes a dual-function enzyme called catalase-peroxidase, which confers sensitivity in M. tuberculosis to isonicotinic acid hydrazide. We have constructed a system for the high level expression of a recombinant form of this enzyme by amplifying the katGgene from the pYZ56 construct (1) and subcloning into a vector suitable for expression in Escherichia coli. The resulting plasmid, pTBCP, produced the catalase-peroxidase in large quantities, corresponding to 30% of total cell protein. The enzyme has been purified to homogeneity and appears to be a dimer in the native form. Using either hydrogen peroxide or t-butyl hydroperoxide and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) as substrates,k cat and K m values have been obtained for both catalatic and peroxidatic activities, respectively. The availability of significant quantities of an active, folded, recombinant form of M. tuberculosiscatalase-peroxidase should thus facilitate future studies of its role in drug activation and antibiotic resistance.

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.

Comparison of two combinations of cyanine dyes for prelabelling and gel electrophoresis.

We report the use of IC‐OSu ethyl‐Cy3 and ethyl‐Cy5 N‐hydroxysuccinimide ester (NHS) cyanine dyes, which have similar chemical properties as the CyDye™ DIGE fluor minimal dyes for pre‐electrophoresis labelling. Multiple sample analyses in different laboratories indicate that the use of IC‐OSu ethyl‐Cy3 and ethyl‐Cy5 NHS ester cyanine dyes produces equivalent results to those obtained with DIGE CyDyes, and allows sample multiplexing and accurate quantitation for differential proteome analysis.Schizophrenia is a debilitating condition associated with high morbidity and mortality. While currently available atypical antipsychotic agents have significantly advanced the treatment of schizophrenia, there is still a great unmet need for new, effective and better-tolerated therapies. Iloperidone, a D(2)/5-HT(2) receptor antagonist, has been recently approved by the US FDA for the acute treatment of schizophrenia in adults. Iloperidone has been shown to be effective in the treatment of schizophrenia in four short-term (4-6 weeks) and three long-term (52 weeks) studies with over 3000 patients exposed to treatment. Results also indicated a reassuring safety profile, particularly regarding extrapyramidal symptoms, akathisia and prolactin elevation, with a modest effect on weight gain and no medically important changes in cholesterol, triglycerides and glucose. As other antipsychotics, iloperidone has been shown to prolong the QTc interval. Since none of the current therapies work for every patient, a pharmacogenetic approach was used to identify genetic markers associated with increased response to iloperidone, suggesting a personalized therapeutic option for this drug. In addition, a long-term 4-week injectable formulation is being developed that may assist with patient compliance. Key development findings for iloperidone are presented here.

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.

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.

Mycobacterium tuberculosis antigen 85B and ESAT-6 expressed as a recombinant fusion protein in Mycobacterium smegmatis elicits cell-mediated immune response in a murine vaccination model

In this study, we investigated the potential molecular and immunological differences of a recombinant fusion protein (Hybrid-1), comprising of the immunodominant antigens Ag85B and ESAT-6 from Mycobacterium tuberculosis, derived from two different expression systems, namely Mycobacterium smegmatis and Escherichia coli. The fusion protein was successfully expressed and purified from both bacterial hosts and analyzed for any host-dependent post-translational modifications that might affect the immunogenicity of the protein. We investigated the immunogenicity of Hybrid-1 expressed in the two host species in a murine vaccination model, together with a reference standard Hybrid-1 (expressed in E. coli) from the Statens Serum Institut. No evidence of any post-translation modification was found in the M. smegmatis-derived Hybrid-1 fusion protein, nor were there any significant differences in the T-cell responses obtained to the three antigens analyzed. In conclusion, the Hybrid-1 fusion protein was successfully expressed in a homologous expression system using M. smegmatis and this system is worth considering as a primary source for vaccination trials, as it provided protein of excellent yield, stability and free from lipopolysaccharide.