Taurine, a Naturally Occurring Amino Acid, as a Physical Stability Enhancer of Different Monoclonal Antibodies.

Degradation of therapeutic monoclonal antibodies (mAbs) is a major concern as it affects efficacy, shelf-life, and safety of the product. Taurine, a naturally occurring amino acid, is investigated in this study as a potential mAb stabilizer with an extensive analytical characterization to monitor product degradation. Forced degradation of trastuzumab biosimilar (mAb1)-containing samples by thermal stress for 30 min resulted in high-molecular-weight species by more than 65% in sample without taurine compared to the sample with taurine. Samples containing mAb1 without taurine also resulted in higher Z-average diameter, altered protein structure, higher hydrophobicity, and lower melting temperature compared to samples with taurine. The stabilizing effect of taurine was retained at different mAb and taurine concentrations, time, temperatures, and buffers, and at the presence of polysorbate 80 (PS80). Even the lowest taurine concentration (10 mM) considered in this study, which is in the range of taurine levels in amino acid injections, resulted in enhanced mAb stability. Taurine-containing samples resulted in 90% less hemolysis than samples containing PS80. Additionally, mAb in the presence of taurine showed enhanced stability upon subjecting to stress with light of 365 nm wavelength, combination of light and H2O2, and combination of Fe2+ and H2O2, as samples containing mAb without taurine resulted in increased degradation products by more than 50% compared to samples with taurine upon subjecting to these stresses for 60 min. In conclusion, the presence of taurine enhanced physical stability of mAb by preventing aggregate formation, and the industry can consider it as a new mAb stabilizer.

Does Aggregation of Therapeutic IgGs in PBS Offer a True Picture of What Happens in Models Derived from Human Body Fluids?

Therapeutic proteins such as monoclonal antibodies (mAb) are known to form aggregates due to various factors. Phosphate buffered saline (PBS), human serum, and human serum filtrate (HSF) are some of the models used to analyze mAb stability in physiologically relevant in-vitro conditions. In this study, aggregation of mAb in PBS and models derived from body fluids seeded with mAb samples subjected to various stresses were compared. Samples containing mAb subjected to pH, temperature, UV light, stirring, and interfacial agitation stress were seeded into different models for 2 case studies. In the first case study, %HMW (high molecular weight species) of mAb in PBS and HSF were compared using size exclusion chromatography. It was found that change in %HMW was higher in PBS compared to HSF. For example, PBS containing mAb that was subjected to UV light stress showed change in HMW by >10 % over 72 h, but the change was <5 % in HSF. In second case study, aggregates particles of FITC tagged mAb were monitored in PBS and serum using fluorescence microscope image processing. It was found that PBS and serum containing mAb subjected to stirring and interfacial agitation resulted in aggregates of >2 µm size, and average size and percentage number of particles having >10 µm size was higher in serum compared to PBS at all analysis time point. Overall, it was found that aggregation of mAb in PBS was different from that in human body fluids. Second case study also showed the importance of advanced strategies for further characterization of mAb in serum.

Combined Presence of Ferrous Ions and Hydrogen Peroxide in Normal Saline and In Vitro Models Induces Enhanced Aggregation of Therapeutic IgG due to Hydroxyl Radicals.

Therapeutic monoclonal antibodies (mAb) are known to form aggregates and fragments upon exposure to hydrogen peroxide (H2O2) and ferrous ions (Fe2+). H2O2 and Fe2+ react to form hydroxyl radicals that are detrimental to protein structures. In this study, aggregation of mAb in the combined presence of Fe2+ and H2O2 was investigated in saline and physiologically relevant in vitro models. In the first case study, forced degradation of mAb in saline (a fluid used for administration of mAb) was carried out at 55 °C in the combined presence of 0.2 mM Fe2+ and 0.1% H2O2. The control and stressed samples were analyzed using an array of techniques including visual observation, size-exclusion chromatography (SEC), dynamic light scattering (DLS), microscopy, UV-vis, fluorescence, Fourier transform infrared spectroscopy, and cell-based toxicity assays. At the end of 1 h, samples having the combined presence of both Fe2+ and H2O2 exhibited more than 20% HMW (high molecular weight species), whereas samples having only Fe2+, H2O2, or neither resulted in less than 3% HMW. Aggregate-rich samples also exhibited altered protein structures and hydrophobicity. Aggregation increased upon increasing the time, temperature, and concentration of Fe2+ and H2O2. Samples having both Fe2+ and H2O2 also showed higher cytotoxicity in red blood cells. Samples of mAb with chlorides of copper and cobalt with H2O2 also resulted in multifold degradation. The first case study showed enhanced aggregation of mAb in the combined presence of Fe2+ and H2O2 in saline. In the second case study, aggregation of mAb was investigated in artificially prepared extracellular saline and in vitro models such as macromolecule free fraction of serum and serum. In the presence of both Fe2+ and H2O2, %HMW was higher in extracellular saline compared to macromolecule free fraction of serum. Further, in vitro models having the combined presence of Fe2+ and H2O2 resulted in enhanced aggregation of mAb compared to models that had neither.

A polymeric Brønsted acid ionic liquid mediated liquefaction of municipal solid waste.

The rapid industrialization and population explosion continuously generate massive amounts of municipal waste. Several conventional processes are in practice for the treatment of municipal waste, but the requirement of stringent operating conditions, incomplete conversion, longer processing time and emission of toxic gases, etc., are the major associated barriers. Thus, there is an urgent requirement for a sustainable, environmentally feasible process that can process waste into energy and fuel products. In the present manuscript, polyethylenimine functionalized polymeric Bronsted acid ionic liquid (PolyE-IL) catalysts have been explored for the Catalytic Thermo Liquefaction (CTL) of organic biodegradable municipal solid waste (MSW). A series of PolyE-IL catalysts with variable counter ions were examined for CTL of MSW. Of all the tested PolyE-IL catalysts, the integration of [PEI]+[HSO4]- gave excellent MSW conversion (>85%) and yield (>80%) of liquefied products (CTL-Oil) under non-stringent reaction conditions and without any formation char and gases. The influence of reaction conditions such as catalyst concentration, reaction temperature, time, slurry concentration, and type of feedstock of conversion and yield are studied. The column adsorption and membrane separation process was integrated to facilitate the catalyst and CTL-Oil separation. A series of commercially available hydrophobic resins were tested to separate catalyst and CTL-Oil. ICT005 showed the highest adsorption efficiency of all tested resins with 35.46 mg/mL of binding capacity and Kd of 0.02159. The physicochemical properties of CTL-Oil were studied in detail by using various analytical tools, which exhibited that CTL-Oil comprises a mixture of small and large molecular weight organic compounds and has a calorific value of 4000 kcal/kg; hence it could be used for further energy and fuel applications. Thus, the reported CTL process can be beneficial to resolve both environmental and fossil fuel dependency issues simultaneously by converting MSW into CTL-Oil.

Image Analysis Algorithm-Based Platform for Determining Micron and Higher Aggregate Size Distribution of Therapeutic IgG Using Brightfield and Fluorescence Microscope Images.

A platform for determining size distribution of micron (1-100 µm) and larger (> 100 µm) aggregates of therapeutic IgG has been established by using image processing algorithms for brightfield and fluorescence microscope images. The algorithm for brightfield images involved conversion to grayscale followed by pixel-based and size-based thresholding. Morphological operations were then applied and the size distribution of aggregates were extracted. Fluorescence images of the aggregates of mAb tagged by a fluorescent dye were captured using widefield fluorescence microscope, confocal laser scanning microscope, and Cytell Cell Imaging System and the images were processed using a series of denoising steps followed by thresholding and morphological operations. The samples were subjected to different stresses, among which the aggregates were visible in the microscope for sample subjected to bubbling, stirring, and temperature. The images of these aggregates were effectively denoised and the size distribution of aggregates was analyzed using the algorithm. The overall aggregate size distribution obtained by image processing ranged in the micron and higher size range. The size obtained from brightfield image processing was validated using images of liquid chromatography resins. Further, the aggregate size distribution obtained using image processing was compared with experimental techniques such as Mastersizer 2000 and Micro Flow Imaging. It was found that analysis of IgG aggregates using image processing could serve as an orthogonal methodology to the existing approaches.

Rapid aggregation of therapeutic monoclonal antibodies by bubbling induced air/liquid interfacial and agitation stress at different conditions.

Degradation of therapeutic monoclonal antibodies (mAb) due to interfacial agitation through air bubbling was investigated. Samples containing mAb in phosphate buffered saline were subjected to rapid bubbling by using a peristaltic pump at an air flow rate of 11.5 mL/min. Samples were analyzed by visual observation, UV-Vis, fluorescence, circular dichroism and infrared spectroscopy, size-exclusion chromatography (SEC), dynamic light scattering, microscopy, and cell-based activity assays. The stressed samples showed increasing turbidity with bubbling time, with mAb1 showing a protein loss of 53% in the supernatant at the latest time point (240 min), indicating formation of sub-visible and visible aggregates. Aggregate rich samples exhibited altered secondary structure and higher hydrophobicity with 40% reduction in activity. The supernatants of the stressed samples showed unchanged secondary and tertiary structure without the presence of any oligomers in SEC. Furthermore, the impact of various factors that could affect aggregation was investigated and it was found that the extent of aggregation was affected by protein concentration, sample volume, presence of surfactants, temperature, air flow rate, and presence of silicone oil. In conclusion, exposure to air/liquid interfacial stress through bubbling into liquid mAb samples effectively generated sub-visible and visible aggregates, making air bubbling an attractive approach for interfacial stress degradation studies of mAbs.

Novel semi-automated fluorescence microscope imaging algorithm for monitoring IgG aggregates in serum.

Analysis of therapeutic IgG aggregates in serum is a potential area of investigation as it can give deeper insights about the function, immunogenic issues and protein interaction associated with the aggregates. To overcome various complexities associated with the existing analytical techniques for analyzing aggregates in serum, a novel florescence microscopy-based image processing approach was developed. The monoclonal antibody (mAb) was tagged with a fluorescent dye, fluorescein isothiocyanate (FITC). Aggregates, generated by stirring, were spiked into serum and images were captured at various time points. After denoising, thresholding by weighted median, 1D Otsu, and 2D Otsu was attempted and a modified 2D Otsu, a new mode of thresholding, was developed. This thresholding method was found to be highly effective in removing noises and retaining analyte sizes. Out of 0-255, the optimized threshold value obtained for the images discussed in modified 2D Otsu was 9 while 2D Otsu's overestimated values were 38 and 48. Other morphological operations were applied after thresholding and the area, perimeter, circularity, and radii of the aggregates in these images were calculated. The proposed algorithm offers an approach for analysis of aggregates in serum that is simpler to implement and is complementary to existing approaches.

BAILs mediated Catalytic Thermo Liquefaction (CTL) process to convert municipal solid waste into carbon densified liquid (CTL-Oil).

Continual increase in municipal solid waste (MSW) posing global environmental challenge which directed focus towards the waste to energy to achieve dual goal of waste minimization and energy generation. The present manuscript introducing Bronsted acid ionic liquids (BAILs) mediated Catalytic Thermo Liquefaction (CTL) process for conversion of MSW into carbon densified liquid (CTL-Oil) which can be used for multiple energy and fuel applications. BAILs with different counter ions were synthesized and tested for CTL of wet organic biodegradable MSW. The exploration of BAILs provides significant benefits in terms of operating conditions (120 °C, 90 min) with zero char and gases. Of the synthesized catalysts [Benz-SO3HIm]+[H2PO4]-,[Benz-SO3Him]+[HSO4]-,[Benz-SO3HIm]+[TsO]-and [BenzSO3HIm]+[TfO]-, BAIL with [HSO4]-counter ion showed a profound effect on CTL. The intensified CTL process resulted in > 85% MSW conversion with > 80% yield of CTL-Oil without any char and gas formation. Use of BAILs assisted the ease of dissolution and hydrolysis of biomass to produce CTL-Oil via hydrolysis, condensation, cyclization and dehydration reactions. The plausible mechanism for CTL has been proposed. The physicochemical analysis of CTL-Oil was conducted by using elemental analysis, Bomb calorimeter, GC-MS and ATR-FTIR. It was found that the CTL-Oil was rich source of C (48-55%), H (6-8%), O (30-41%) containing compounds such as long-chain hydrocarbons, carboxylic acids, heterocyclic compounds, aldehydes, ketones and esters, etc. Furthermore, the calorific value of CTL-Oil was found to be 20-23 MJ/kg, thus it can be explored for multiple energy and fuel applications. However, the CTL process also adds several environmental and process economic benefits over the conventional waste liquefaction/disposal processes.

Structure-Based Design of Small Peptide Ligands to Inhibit Early-Stage Protein Aggregation Nucleation.

We report a structure-based approach to design peptides that can bind to aggregation-prone, partially folded intermediates (PFI) of insulin, thereby inhibiting early stages of aggregation nucleation. We account for the important role of the modular architecture of protein-protein binding interfaces and tertiary structure heterogeneity of the PFIs in the design of peptide inhibitors. The determination of association hotspots revealed that two interface segments are required to capture majority contribution to insulin homodimer binding energy. The selection of peptides that will have a high probability to inhibit insulin self-association was done on the basis of similarity in binding interface coverage of PFI residues in the peptide-PFI complex and the native-PFI dimer. Data on aggregate growth rate and secondary structure for formulations incubated under amyloidogenic conditions show that designed peptides inhibit insulin aggregation in a concentration-dependent manner. The mechanism of aggregation inhibition was probed by determining the enthalpy of peptide-insulin binding and peptide micellization using isothermal titration calorimetry. Finally, the effect of designed peptides on insulin activity was quantified using a spectrophotometric assay for glucose uptake by HepG2 cells.

Does interaction of monoclonal antibody charge variants with VEGF-A and ELISA reagents affect its quantification?

The ability of an enzyme linked immunosorbent assay (ELISA) to measure the concentration of charge variants of a monoclonal antibody (mAb) biotherapeutic has been investigated. Percentage recovery was found to be in the range of 80%-120% with inter and intra assay variation between 5% and 15%. Linear regression analysis of ELISA output at different dilutions yielded a good fit (R2 > 0.98). Assay output was not hindered by the presence of serum proteins and other analytes such as GCSF, demonstrating acceptable precision and specificity. It was concluded that the charge variants of the mAb can be accurately quantified by ELISA.