Characterisation of Stress-Induced Aggregate Size Distributions and Morphological Changes of a Bi-Specific Antibody Using Orthogonal Techniques.

A critical step in monoclonal antibody (mAb) screening and formulation selection is the ability of the mAb to resist aggregation following exposure to environmental stresses. Regulatory authorities welcome not only information on the presence of micron-sized particles, but often any information on sub-visible particles in the size range obtained by orthogonal sizing techniques. The present study demonstrates the power of combining established techniques such as dynamic light scattering (DLS) and micro-flow imaging (MFI), with novel analyses such as raster image correlation spectroscopy (RICS) that offer to bridge existent particle sizing gaps in this area. The influence of thermal and freeze-thaw stress treatments on particle size and morphology was assessed for a bi-specific antibody (mAb2). Aggregation of mAb2 was confirmed to be concentration- and treatment-dependent following thermal stress and freeze-thaw cycling. Particle size and count data show concentration- and treatment-dependent behaviour of aggregate counts, morphological descriptors and particle size distributions. Complementarity in particle size output was observed between all approaches utilised, where RICS bridged the analytical size gap (∼0.5-5 µm) between DLS and MFI. Overall, this study highlights the potential of orthogonal image analyses such as RICS (analytical size gap) and MFI (particle morphology) for formulation screening.

Taking your breath away: metabolomics breathes life in to personalized medicine.

Breath-based metabolomics (breathomics) is an exciting developing area of biotechnology that centers on the capture, identification, and quantification of volatile organic compound (VOC) patterns in human breath and their utilization as tools in the diagnosis of a broad spectrum of medical problems. With the age of personalized medicines demanding rapid bespoke diagnosis and treatment, this area of molecular diagnostics is beginning to see an upsurge in biotechnological advancement. Here, we discuss recent improvements and directions in the development of breath VOC analysis and diagnosis platforms that offer the potential for disease biomarker discovery and disease prognosis.

Quantitative assessment of p-glycoprotein expression and function using confocal image analysis.

P-glycoprotein is implicated in clinical drug resistance; thus, rapid quantitative analysis of its expression and activity is of paramout importance to the design and success of novel therapeutics. The scope for the application of quantitative imaging and image analysis tools in this field is reported here at "proof of concept" level. P-glycoprotein expression was utilized as a model for quantitative immunofluorescence and subsequent spatial intensity distribution analysis (SpIDA). Following expression studies, p-glycoprotein inhibition as a function of verapamil concentration was assessed in two cell lines using live cell imaging of intracellular Calcein retention and a routine monolayer fluorescence assay. Intercellular and sub-cellular distributions in the expression of the p-glycoprotein transporter between parent and MDR1-transfected Madin-Derby Canine Kidney cell lines were examined. We have demonstrated that quantitative imaging can provide dose-response parameters while permitting direct microscopic analysis of intracellular fluorophore distributions in live and fixed samples. Analysis with SpIDA offers the ability to detect heterogeniety in the distribution of labeled species, and in conjunction with live cell imaging and immunofluorescence staining may be applied to the determination of pharmacological parameters or analysis of biopsies providing a rapid prognostic tool.

Monitoring the kinetics of CellTrace™ calcein red-orange AM intracellular accumulation with spatial intensity distribution analysis.

BACKGROUND: Routine black box approaches quantify fluorescence intensity to profile the uptake of fluorophores, providing limited insight into microscopic events. Spatial intensity distribution analysis has previously been reported to quantify oligomerisation and number of particles from selected regions and profile intracellular distributions of labelled moieties. METHODS: In this study, the concentration and time-dependent behaviour of CellTrace™ calcein red-orange (AM) intracellular accumulation was examined in colorectal adenocarcinoma cell line and bovine aortic endothelial cells. Monolayers were subjected to fluorescence correlation spectroscopy, fluorescence intensity and SpIDA measurements to determine differences in the rate and extent of intracellular accumulation. RESULTS: Intracellular accumulation data derived from Spatial intensity distribution analysis were found to correlate with that of fluorescence correlation spectroscopy and fluorescence intensity profiles. The extent of intracellular accumulation was found to be time and concentration-dependent in both cell lines examined, with no significant differences in the rate of intracellular accumulation. CONCLUSIONS: Spatial intensity distribution analysis applied at 'proof of concept' level is a rapid and user-friendly tool that can be applied to the quantification of intracellular concentration and kinetics of fluorophore uptake. GENERAL SIGNIFICANCE: Confocal imaging as a routinely implemented tool for profiling fluorescently-labelled species is often under-exploited for yielding quantitative parameters.

Real-time evaluation of aggregation using confocal imaging and image analysis tools.

Real-time confocal imaging was utilised to monitor the in situ loss of BSA monomers and aggregate formation using Spatial Intensity Distribution Analysis (SpIDA) and Raster Image Correlation Spectroscopy (RICS). At the proof of concept level this work has demonstrated the applicability of RICS and SpIDA for monitoring protein oligomerisation and larger aggregate formation.

On the cellular uptake and membrane effect of the multifunctional peptide, TatLK15.

Recently, the multifunctional peptide TatLK15 resulting from the fusion of the cell penetrating peptide Tat and the amphipathic peptide LK15 was shown to be efficient at mediating siRNA and shRNA delivery in leukemia cells to silence the bcr-abl oncoprotein. The present study focused on TatLK15 peptide cellular uptake and defining conditions for its use within a range of doses exhibiting minimal toxicity. The initial part of the study carried out in solution confirmed that the insertion of a glycine bridge allowed retention of the LK15 α-helicity, and fluorescence correlation spectroscopy did not reveal preferential conformations at the studied concentrations. In the second part, TatLK15 uptake mechanisms appeared peptide dose- and cell line- dependent as well as requiring membrane potential. Below a critical dose, TatLK15 toxicity appeared limited for approximately three hours as demonstrated by the combined use of lactate dehydrogenase release, MTT assays, and time-dependent observation of membrane-impermeant dye uptake using high content screening apparatus. Furthermore, toxicity was observed to occur rapidly at higher peptide doses. Finally, a comparison between TatLK15 and another Tat amphipathic peptide construct suggested that α-helix content should be viewed as a key element in the development of similar peptides.

Proteins behaving badly: emerging technologies in profiling biopharmaceutical aggregation.

Over recent decades biotechnology has made significant advances owing to the emergence of powerful biochemical and biophysical instrumentation. The development of such technologies has enabled high-throughput assessment of compounds, the implementation of recombinant DNA technology, and large-scale manufacture of monoclonal antibodies. Such innovations have ultimately resulted in the current experienced biopharmaceutical stronghold in the therapeutic market. Yet aggregate prediction and profiling remains a challenge in the formulation of biopharmaceuticals due to artifacts associated with each analytical method. We review some emerging trends and novel technologies that offer a promising potential for accurately predicting and profiling protein aggregation at various stages of biopharmaceutical product design.

Raster image correlation spectroscopy as a novel tool for the quantitative assessment of protein diffusional behaviour in solution.

The application of raster image correlation spectroscopy (RICS) as a tool for the characterisation of protein diffusion was assessed using a model protein, bovine serum albumin (BSA), as a function of formulation and denaturing conditions. RICS results were also validated against dynamic light scattering and fluorescence correlation spectroscopy. Results from this study demonstrate correlation between outputs obtained from the three experimental techniques. Ionic strength independency was observed at pH 7, and a reduction in the corresponding diffusion coefficients was noted at pH 4.5 for 1 µM BSA-Alexa Fluor 488. Conversely, at pH 5.2, higher-concentration samples exhibited ionic strength dependency. Buffer composition, sample pretreatment, thermal denaturation and freeze-thaw cycling were also found to influence RICS output, with a reduction in the diffusion coefficient and the number of particles observed for both pH values. In conclusion, RICS analysis of images acquired using a commercial confocal microscope offers a potential scope for application to both quantitative and qualitative characterisation of macromolecular behaviour in solution.