Development of hydrolytic stability screening methods for early drug discovery with high differentiation and predictive power.

In early drug discovery, hydrolytic chemical stability is routinely assessed to ensure future developability of quality compounds and stability in in vitro test environments. When conducting high-throughput hydrolytic stability analyses as part of the compound risk assessment, aggressive conditions are typically applied to allow for faster screening. However, it can be challenging to extrapolate the real stability risk and to rank compounds due to over-estimating risk based on aggressive conditions and the narrow discriminative window. In this study, critical assay parameters including temperature, concentration, and detection technique were systematically assessed using selected model compounds, and the impact and interplay of these parameters on predictive power and prediction quality were evaluated. Improved data quality was achieved using high sample concentration and reduced temperature, combined with ultraviolet (UV) detection, while mass spectrometry (MS) detection was found to be a useful complementary detection technique. Therefore, a highly discriminative stability protocol with optimized assay parameters and experimental data quality is proposed. The optimized assay can provide early guidance on the potential stability risk of a drug molecule as well as enable more confident decision-making in compound design, selection, and development.

The Degradation Map Process - A Tool for Obtaining a Lean Stability Strategy in Drug Development.

Stability is fundamental when exploring a drug candidate's potential as a drug product. During the pharmaceutical industry drug development process information regarding stability and degradation are captured in different departments, e.g. from discovery to operations, and will be included in the overall control strategy. With a profound understanding of a drug candidate's degradation chemistry, a science and risk based approach in progressing a lean stability strategy is possible. This case study present a clear and visible concept to facilitate a lean stability strategy by the use of degradation maps and describes a process for how these can be used during drug development. The understanding of possible and/or observed degradation pathways will guide the design of the drug product and stability studies in development. A degradation map displays degradation pathways with short comments on the reaction/mechanism involved. The degradation map process starts with a theoretical degradation map. The map is updated as the drug project progresses, preferably after forced degradation experiments, after compatibility studies and finally when the late stage formulation is set. The degradation map should be used to capture information of intrinsic chemical properties of the active pharmaceutical ingredient (API) and can thereby be used to mitigate stability issues. The map is foremost a cross-functionally available tool collecting and visualizing stability information throughout the development process, and as such a valuable tool to efficiently develop a lean stability strategy.

Characterization and demonstration of drug compound ring-chain tautomer formation and its impacts on quality control.

Unknown chromatographic peaks, potential impurities, were observed in a series of related compounds. This led to the identification and characterization of tautomeric equilibria. Structural elucidation was required to understand the potential impurity profile, thus impacting method development for quality control. In this work, characterization of the chemical structures, AZ13581258 and AZD5718, and equilibria of the tautomeric forms was performed using a range of advanced analytical techniques such as preparative chromatography, nuclear magnetic resonance (NMR), chromatographic detection by mass spectrometry (MS), MSMS, and ultraviolet spectroscopy (UV). Predictions using density functional theory (DFT) further explains and confirms the tautomer equilibria through predictions of reaction barrier energies, UV-spectra and NMR data. These investigations led to fully understand the impurity profile and to the development of a quality control method for AZD5718 drug substance and drug product. In conclusion, ring-chain tautomeric structures are predominately formed under acidic conditions, and the additional peaks observed in LC during organic impurity determination were found to originate from ring-chain closed tautomers in equilibria with the parent open form compound. Hence, the closed and open tautomer forms should all be considered as the same compound.

Qualification of impurities based on metabolite data.

Regulatory Guidance documents ICH Q3A (R2) and ICH Q3B (R2) state that "impurities that are also significant metabolites present in animal and/or human studies are generally considered qualified". However, no guidance is provided regarding data requirements for qualification, nor is a definition of the term "significant metabolite" provided. An opportunity is provided to define those categories and potentially avoid separate toxicity studies to qualify impurities. This can reduce cost, animal use and time, and avoid delays in drug development progression. If the concentration or amount of a metabolite, in animals or human, is similar to that of the known, structurally identical impurity (arising from the administered test material), the qualification of the impurity on the grounds of it also being a metabolite is justified. We propose two complementary approaches to support conclusions to this effect: 1) demonstrate that the impurity is formed by metabolism in animals and/or man, based preferably on plasma exposures or, alternatively, amounts excreted in urine, and, where appropriate, 2) show that animal exposure to (or amount of) the impurity/metabolite is equal or greater in animals than in humans. An important factor of both assessments is the maximum theoretical concentration (or amount) (MTC or MTA) of the impurity/metabolite achievable from the administered dose and recommendations on the estimation of the MTC and MTA are presented.

Experimental and Quantum Chemical Evaluations of Pyridine Oxidation Under Drug Development Stress Test Conditions.

The oxidation reaction of pyridine by hydrogen peroxides in water media was investigated by combining quantum chemical calculations and laboratory experiments. Pyridine was selected as a model system for aromatic amines that frequently occurs in drug molecules. Several different reaction conditions, commonly used in stress testing of drug molecules during drug development, were investigated to increase mechanistic insight to this class of oxidation reactions. Of special interest is to note that small amounts of acetonitrile, a regularly used cosolvent to keep poorly soluble drug molecules in water solution, could catalyze the oxidation reaction in the presence of hydrogen peroxide. Consequently, attention needs to be taken when comparing data from different stress test studies of amine oxidation by hydrogen peroxides at different pH, and with and without acetonitrile. In particular, they need to be controlled when identifying the proper intrinsic stability of the drug molecule.

Parallel production and verification of protein products using a novel high-throughput screening method.

Protein production and analysis in a parallel fashion is today applied in laboratories worldwide and there is a great need to improve the techniques and systems used for this purpose. In order to save time and money, a fast and reliable screening method for analysis of protein production and also verification of the protein product is desired. Here, a micro-scale protocol for the parallel production and screening of 96 proteins in plate format is described. Protein capture was achieved using immobilized metal affinity chromatography and the product was verified using matrix-assisted laser desorption ionization time-of-flight MS. In order to obtain sufficiently high cell densities and product yield in the small-volume cultivations, the EnBase® cultivation technology was applied, which enables cultivation in as small volumes as 150 µL. Here, the efficiency of the method is demonstrated by producing 96 human, recombinant proteins, both in micro-scale and using a standard full-scale protocol and comparing the results in regard to both protein identity and sample purity. The results obtained are highly comparable to those acquired through employing standard full-scale purification protocols, thus validating this method as a successful initial screening step before protein production at a larger scale.

Enhancing the protein production levels in Escherichia coli with a strong promoter.

In biotechnology, the use of Escherichia coli for recombinant protein production has a long tradition, although the optimal production conditions for certain proteins are still not evident. The most favorable conditions for protein production vary with the gene product. Temperature and induction conditions represent parameters that affect total protein production, as well as the amount of soluble protein. Furthermore, the choice of promoter and bacterial strain will have large effects on the production of the target protein. In the present study, the effects of three different promoters (T7, trc and lacUV5) on E. coli production of target proteins with different characteristics are presented. The total amount of target protein as well as the amount of soluble protein were analyzed, demonstrating the benefits of using a strong promoter such as T7. To understand the underlying causes, transcription levels have been correlated with the total amount of target protein and protein solubility in vitro has been correlated with the amount of soluble protein that is produced. In addition, the effects of two different E. coli strains, BL21(DE3) and Rosetta(DE3), on the expression pattern were analyzed. It is concluded that the regulation of protein production is a combination of the transcription and translation efficiencies. Other important parameters include the nucleotide-sequence itself and the solubility of the target protein.

Surrogate antigens as targets for proteome-wide binder selection.

In the past decade, many initiatives were taken for the development of antibodies for proteome-wide studies, as well as characterisation and validation of clinically relevant disease biomarkers. Phage display offers many advantages compared to antibody generation by immunisation because it is an unlimited resource of affinity reagents without batch-to-batch variation and is also amendable for high throughput in contrast to conventional hybridoma technology. One of the major bottlenecks to proteome-wide binder selection is the limited supply of suitable target antigens representative of the human proteome. Here, we provide proof of principle of using easily accessible, cancer-associated protein epitope signature tags (PrESTs), routinely generated within the Human Protein Atlas project, as surrogate antigens for full-length proteins in phage selections for the retrieval of target-specific binders. These binders were subsequently tested in western blot, immunohistochemistry and protein microarray application to demonstrate their functionality.

Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3).

The effect of two Escherichiacoli expression strains on the production of recombinant human protein fragments was evaluated. High-throughput protein production projects, such as the Swedish Human Protein Atlas project, are dependent on high protein yield and purity. By changing strain from E. coli BL21(DE3) to E. coli Rosetta(DE3) the overall success rate of the protein production has increased dramatically. The Rosetta(DE3) strain compensates for a number of rare codons. Here, we describe how the protein expression of human gene fragments in E. coli strains BL21(DE3) and Rosetta(DE3) was evaluated in two stages. Initially a test set of 68 recombinant proteins that previously had been expressed in BL21(DE3) was retransformed and expressed in Rosetta(DE3). The test set generated very positive results with an improved expression yield and a significantly better purity of the protein product which prompted us to implement the Rosetta(DE3) strain in the high-throughput protein production. Except for analysis of protein yield and purity the sequences were also analyzed regarding number of rare codons and rare codon clusters. The content of rare codons showed to have a significant effect on the protein purity. Based on the results of this study the atlas project permanently changed expression strain to Rosetta(DE3).

A global view of protein expression in human cells, tissues, and organs.

Defining the protein profiles of tissues and organs is critical to understanding the unique characteristics of the various cell types in the human body. In this study, we report on an anatomically comprehensive analysis of 4842 protein profiles in 48 human tissues and 45 human cell lines. A detailed analysis of over 2 million manually annotated, high-resolution, immunohistochemistry-based images showed a high fraction (>65%) of expressed proteins in most cells and tissues, with very few proteins (<2%) detected in any single cell type. Similarly, confocal microscopy in three human cell lines detected expression of more than 70% of the analyzed proteins. Despite this ubiquitous expression, hierarchical clustering analysis, based on global protein expression patterns, shows that the analyzed cells can be still subdivided into groups according to the current concepts of histology and cellular differentiation. This study suggests that tissue specificity is achieved by precise regulation of protein levels in space and time, and that different tissues in the body acquire their unique characteristics by controlling not which proteins are expressed but how much of each is produced.

Automated sample preparation method for mass spectrometry analysis on recombinant proteins.

A completely automated procedure for the purification and desalting of proteins with a polyhistidine purification tag prior to mass spectrometry analysis is presented. The system is ideal for rapid quality control and optimization studies and it provides researchers with a straightforward, reliable tool for studies of recombinant proteins. Forty-eight samples can be prepared within 4.5h and only small cultivation and buffer volumes are needed. In this proof of concept, 19,000-35,000Da recombinant proteins from both crude and clarified cell lysates were successfully prepared for subsequent analysis by electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry as well as by gel electrophoresis.

High-throughput protein production--lessons from scaling up from 10 to 288 recombinant proteins per week.

The demand for high-throughput recombinant protein production has markedly increased with the increased activity in the field of proteomics. Within the Human Protein Atlas project recombinantly produced human protein fragments are used for antibody production. Here we describe how the protein expression and purification protocol has been optimized in the project to allow for handling of nearly 300 different proteins per week. The number of manual handling steps has been significantly reduced (from 18 to 9) and the protein purification has been completely automated.

A genecentric Human Protein Atlas for expression profiles based on antibodies.

An attractive path forward in proteomics is to experimentally annotate the human protein complement of the genome in a genecentric manner. Using antibodies, it might be possible to design protein-specific probes for a representative protein from every protein-coding gene and to subsequently use the antibodies for systematical analysis of cellular distribution and subcellular localization of proteins in normal and disease tissues. A new version (4.0) of the Human Protein Atlas has been developed in a genecentric manner with the inclusion of all human genes and splice variants predicted from genome efforts together with a visualization of each protein with characteristics such as predicted membrane regions, signal peptide, and protein domains and new plots showing the uniqueness (sequence similarity) of every fraction of each protein toward all other human proteins. The new version is based on tissue profiles generated from 6120 antibodies with more than five million immunohistochemistry-based images covering 5067 human genes, corresponding to approximately 25% of the human genome. Version 4.0 includes a putative list of members in various protein classes, both functional classes, such as kinases, transcription factors, G-protein-coupled receptors, etc., and project-related classes, such as candidate genes for cancer or cardiovascular diseases. The exact antigen sequence for the internally generated antibodies has also been released together with a visualization of the application-specific validation performed for each antibody, including a protein array assay, Western blot analysis, immunohistochemistry, and, for a large fraction, immunofluorescence-based confocal microscopy. New search functionalities have been added to allow complex queries regarding protein expression profiles, protein classes, and chromosome location. The new version of the protein atlas thus is a resource for many areas of biomedical research, including protein science and biomarker discovery.

High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography.

A high-throughput protein purification strategy using the polycationic Z(basic) tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Z(basic). These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding pI and solubility, were successfully purified by the laboratory robot. The data show that Z(basic) can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different pI and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His(6)-tag.

A general, robust method for the quality control of intact proteins using LC-ESI-MS.

A simple and robust method for the routine quality control of intact proteins based on liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) is presented. A wide range of prokaryotic and eukaryotic proteins expressed recombinantly in Escherichia coli or Pichia pastoris has been analyzed with medium- to high-throughput with on-line desalting from multi-well sample plates. Particular advantages of the method include fast chromatography and short cycle times, the use of inexpensive trapping/desalting columns, low sample carryover, and the ability to analyze proteins with masses ranging from 5 to 100 kDa with greater than 50 ppm accuracy. Moreover, the method can be readily coupled with optimized chemical reduction and alkylation steps to facilitate the analysis of denatured or incorrectly folded proteins (e.g., recombinant proteins sequestered in E. coli inclusion bodies) bearing cysteine residues, which otherwise form intractable multimers and non-specific adducts by disulfide bond formation.

High-throughput protein purification using an automated set-up for high-yield affinity chromatography.

One of the key steps in high-throughput protein production is protein purification. A newly developed high-yield protein purification and isolation method for laboratory scale use is presented. This procedure allows fully automated purification of up to 60 cell lysates with milligram yields of pure recombinant protein in 18.5h. The method is based on affinity chromatography and has been set up on an instrument that utilizes positive pressure for liquid transfer through columns. A protocol is presented that includes all steps of equilibration of the chromatography resin, load of sample, wash, and elution without any manual handling steps. In contrast to most existing high-throughput protein purification procedures, positive pressure is used for liquid transfer rather than vacuum. Positive pressure and individual pumps for each liquid channel contribute to controlled flow rates and eliminate the risk of introducing air in the chromatography resin and therefore ensure stable chromatography conditions. The procedure is highly reproducible and allows for high protein yield and purity.

Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling.

A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.

High-throughput solubility assay for purified recombinant protein immunogens.

A high-throughput assay is described for analysis of the solubility of purified recombinant proteins. The assay is based on affinity purification of proteins in the presence of chaotropic agents followed by a dilution and incubation step to investigate the solubility in the absence of high concentrations of such agents. The assay can be performed in a 96-well format, which makes it well suited for high-throughput applications. For 125 recombinant proteins expressed as part of an antibody-based proteomics effort, experimental solubility data were compared to calculated hydrophobicity values based on the amino acid sequence of each protein. This comparison showed only weak correlation between the theoretical and experimental values, which emphasizes the importance of experimental assays to determine the solubility of recombinant proteins.

A human protein atlas for normal and cancer tissues based on antibody proteomics.

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

A novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein.

A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow cytometer. Data concerning whole cell fluorescence and light scattering was collected. The whole cell fluorescence is probing intracellular concentrations of soluble fusion proteins. Concurrently, forward scattered light gives data about inclusion body formation, valuable information in process optimisation. To evaluate the method, the cells were disrupted, separated into soluble and non-soluble fractions and analysed by gel electrophoresis. A clear correlation between fluorescence and soluble target protein was shown. Interestingly, the distribution of the cells regarding forward scatter (standard deviation) correlates with the amount of inclusion bodies formed. Finally, the newly developed method was used to evaluate two different purification tags, His(6) and Z(basic), and their effect on the expression pattern.