Analysis of image motion for optical remote sensing satellites staring imaging with area-array detectors.

In this paper, the image motion introduced by the staring action itself during optical remote sensing satellites staring imaging with area-array detectors is studied. The image motion is decomposed into the angle-rotation image motion caused by the change of observation angle, the size-scaling image motion caused by the change of observation distance, and the Earth-rotation image motion caused by the rotation of the ground object with the Earth. The theoretical derivation of the angle-rotation image motion and size-scaling image motion is conducted, and the numerical analysis of the Earth-rotation image motion is carried out. Based on the comparison among the characteristics of the three types of image motions, the conclusion is drawn that, for general staring imaging scenes, the angle-rotation image motion is dominant, followed by the size-scaling image motion and the ignorable Earth-rotation image motion. On the condition that the image motion does not exceed 1 pixel, the allowed maximum exposure time for area-array staring imaging is analyzed. It is found that the large-array satellite is not suitable for long-exposure imaging, as its allowed exposure time decreases rapidly with the increase of roll angle. A satellite with a 12k×12k area-array detector and 500 km orbit is taken as an example. The allowed exposure time is 0.88 s when roll angle of the satellite is 0°; it decreases to 0.02 s when the roll angle increases to 28°.

Enhanced Label-Free Nanoplasmonic Cytokine Detection in SARS-CoV-2 Induced Inflammation Using Rationally Designed Peptide Aptamer.

Rapid and precise serum cytokine quantification provides immense clinical significance in monitoring the immune status of patients in rapidly evolving infectious/inflammatory disorders, examplified by the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. However, real-time information on predictive cytokine biomarkers to guide targetable immune pathways in pathogenic inflammation is critically lacking, because of the insufficient detection range and detection limit in current label-free cytokine immunoassays. In this work, we report a highly sensitive localized surface plasmon resonance imaging (LSPRi) immunoassay for label-free Interleukin 6 (IL-6) detection utilizing rationally designed peptide aptamers as the capture interface. Benefiting from its characteristically smaller dimension and direct functionalization on the sensing surface via Au-S bonding, the peptide-aptamer-based LSPRi immunoassay achieved enhanced label-free serum IL-6 detection with a record-breaking limit of detection down to 4.6 pg/mL, and a wide dynamic range of ∼6 orders of magnitude (values from 4.6 to 1 × 106 pg/mL were observed). The immunoassay was validated in vitro for label-free analysis of SARS-CoV-2 induced inflammation, and further applied in rapid quantification of serum IL-6 profiles in COVID-19 patients. Our peptide aptamer LSPRi immunoassay demonstrates great potency in label-free cytokine detection with unprecedented sensing capability to provide accurate and timely interpretation of the inflammatory status and disease progression, and determination of prognosis.

Identification of Key Excipients for the Solubilization and Structural Characterization of Lipoprotein Lipase, An Enzyme for Hydrolysis of Triglyceride.

Lipoprotein lipase (LPL) is an essential enzyme that hydrolyzes triglycerides in chylomicrons and very low-density lipoprotein into glycerol and fatty acids. One major hurdle in using LPL as a therapeutic has been its poor solubility/stability after purification. Solutions used to preserve purified LPL commonly contain either heparin, or concentrated glycerol and sodium chloride, resulting in hypertonic solutions. These solutions are not acceptable as pharmaceutical formulations. This paper describes the identification of a key excipient, sodium laurate, which can solubilize LPL in an isotonic environment without heparin or concentrated glycerol. A follow-up multi-variant study was performed to identify the effect of sodium laurate and its interaction with sodium chloride on the solubility and processing conditions of LPL. The LPL concentration (up to 14 mg/mL) achievable in pharmaceutically relevant and salt-free conditions was identified to be closely correlated to the concentration of sodium laurate, which was co-concentrated with LPL. The result that sodium laurate increases stability of LPL characterized by differential scanning calorimetry and UV absorbance spectra suggests that the mechanism of solubilization of LPL by sodium laurate is related to LPL structural stabilization. The findings indicate that substrates and their enzymatic products can be strong stabilizers for other protein molecules.

Hydrogen Peroxide Induced Protein Oxidation During Storage and Lyophilization Process.

Although the impact of hydrogen peroxide (HP) on proteins in liquid solutions has been studied extensively, the impact during lyophilization has been largely overlooked. The purpose of this work was to investigate the effect of HP on lyophilized proteins and HP removal by lyophilization. A protein formulation at 5 mg/mL and its placebo were spiked with HP up to 5.0 ppm and then lyophilized. HP concentration, protein oxidation, and aggregation were monitored before and after lyophilization, as well as during storage at 25°C. The lyophilization process removed on average 94.1% of HP from protein formulation, but only 72.5% from the placebo. There were also significant increases in protein oxidization and aggregation. The oxidation increment correlated with the decrease of HP concentration in both the protein formulation and placebo at all temperatures. Protein oxidation at different freezing temperatures was also studied in follow-up studies. Data from these studies suggest that (1) HP has a significant impact on oxidation and aggregation of protein during lyophilization; (2) significant oxidation can occur even when the protein formulation is frozen; (3) the oxidized protein is more prone to aggregation during lyophilization process.

High throughput prediction of the long-term stability of pharmaceutical macromolecules from short-term multi-instrument spectroscopic data.

Changes in the measurements of a macromolecular biopharmaceutical's physical form are often used to predict changes in the drug's long-term stability. These can in turn be used as important markers of changes to a drug's efficacy and safety. Such stability estimates traditionally require human judgment and are frequently tentative. We introduce methods for developing mathematical models that predict a drug's long-term storage stability profile from measurements of short-term physical form and behavior. We measured the long-term (2 year) chemical and colloidal stability of Granulocyte Colony Stimulating Factor (GCSF) in 16 different liquid formulations. Shortly after formulations were placed on stability, we also employed various spectroscopic techniques to characterize the short-term thermal unfolding response of GCSF in the 16 formulations. The short-term data were processed using several data reduction methods, including reduction to spectra at low temperature, to melt curves, and to transition temperatures. Least squares fitting was used to predict the long-term stability measurements from the reduced short-term spectroscopic measurements. On the basis of the cross-validation and a permutation test, many of the long-term stability predictions have less than 1% probability of occurring by chance.

Linking the solution viscosity of an IgG2 monoclonal antibody to its structure as a function of pH and temperature.

Although the viscosity of concentrated antibody solutions has been the focus of many recent studies, less attention has been concentrated on how changes in protein structure impact viscosity. This study examines viscosity profiles of an immunoglobulin G (IgG) 2 monoclonal antibody at 150 mg/mL as a function of temperature and pH. Although the structure of the antibody at pH 4.0-7.0 was comparable at lower temperatures as measured by second derivative UV absorbance and Fourier transform infrared spectroscopy, differences in 8-anilino-1-naphthalene sulfonate (ANS) fluorescence intensity indicated small structural alterations as a function of pH. Below the structural transition onset temperature, the viscosity profiles were pH dependent and linearly correlated with fluorescence intensity, and followed semilogarithmic behavior as a function of temperature. The transitions of the viscosity profiles correlated well with the major structure transitions at a protein concentration of 150 mg/mL. The viscosity correlated particularly well with ANS fluorescence intensity at 0.2 mg/mL below and above the structural transition temperatures. These results suggest: (1) ANS can be an important measure of the overall structure and (2) hydrophobic interactions and charge-charge interactions are the two major physical factors that contribute collectively to the high viscosity of concentrated IgG solutions.

Biophysical and formulation studies of the Schistosoma mansoni TSP-2 extracellular domain recombinant protein, a lead vaccine candidate antigen for intestinal schistosomiasis.

A candidate vaccine to prevent human schistosomiasis is under development. The vaccine is comprised of a recombinant 9 kDa antigen protein corresponding to the large extracellular domain of a tetraspanin surface antigen protein of Schistosoma mansoni, Sm-TSP-2. Here, we describe the biophysical profile of the purified, recombinant Sm-TSP-2 produced in the yeast PichiaPink, which in preclinical studies in mice was shown to be an effective vaccine against intestinal schistosomiasis. Biophysical techniques including circular dichroism, intrinsic and extrinsic fluorescence and light scattering were employed to generate an empirical phase diagram, a color based map of the physical stability of the vaccine antigen over a wide range of temperatures and pH. From these studies a pH range of 6.0-8.0 was determined to be optimal for maintaining the stability and conformation of the protein at temperatures up to 25 °C. Sorbitol, sucrose and trehalose were selected as excipients that prevented physical degradation during storage. The studies described here provide guidance for maximizing the stability of soluble recombinant Sm-TSP-2 in preparation of its further development as a vaccine.

Comparative signature diagrams to evaluate biophysical data for differences in protein structure across various formulations.

A solution to the problem of being able to show statistically significant differences in the measurements of various levels of higher-order protein structure has been an elusive one. We propose the use of comparative signature diagrams (CSDs) to this end. CSDs compare datasets from different biophysical techniques that fingerprint the secondary, tertiary, and quaternary structures of a protein molecule and display statistically significant differences in these datasets. In this paper, we explore the differences in the structures of two proteins (Granulocyte Colony Stimulating Factor [GCSF] and a monoclonal antibody [mAb]) in various formulations. These proteins were chosen based on the extent of differences in structure observed in the formulations. As an initial test, we utilize data from circular dichroism, 8-anilino-1-naphthalene-sulfonate and intrinsic fluorescence spectroscopy, and static light scattering measurements to fingerprint protein structure in the different formulations. Several layers of statistics were explored to visualize the regions of significant differences in the protein spectra. This approach provides a rapid, high-resolution methodology to compare various structural levels of proteins using standard biophysical instrumentation.

Comparison of high-throughput biophysical methods to identify stabilizing excipients for a model IgG2 monoclonal antibody: conformational stability and kinetic aggregation measurements.

The overall conformational stability of a model IgG2 monoclonal antibody (mAb) was examined as a function of temperature and pH using an empirical phase diagram approach. Stabilizing excipients were then identified based on high-throughput methods including (1) kinetic studies measuring aggregation via increases in optical density and (2) thermally induced structural transitions as measured by differential scanning calorimetry (DSC) and fluorescence spectroscopy. The kinetic profiles of antibody aggregation at 65 °C were pH dependent and correlated well with pH effects on secondary and tertiary structural transitions due to heat stress. For the screening of stabilizing excipients, the inhibition of the rate of protein aggregation at pH 4.5 at 65°C, as represented by changes in optical density, was shown to have a clear trend with a modest correlation coefficient compared with the stabilizing effect of the same excipients on the conformational stability of the antibody as measured by DSC and tryptophan fluorescence spectroscopy. These results demonstrate the utility of combining high-throughput data from protein aggregation kinetic experiments and conformational stability studies to identify stabilizing excipients that minimize the physical degradation of an IgG2 mAb.

Design, synthesis, characterization and in-vivo activity of a novel salmon calcitonin conjugate containing a novel PEG-lipid moiety.

OBJECTIVES: The aim of the study was to explore (1) the synthesis of a novel poly(ethylene glycol) modified lipid (PEG-lipid, PL) containing a chemically active tri-block linker, epsilon-maleimido lysine (Mal), and its conjugation with salmon calcitonin (sCT), and (2) the biophysical properties and activity of the resulting conjugate, Mal-PL-sCT, relative to the control, 2PEG-Mal-sCT, which comprises sCT conjugated with alpha-palmitoyl-N-epsilon-maleimido-L-lysine at cysteine 1 and cysteine 7, and PEG moieties at lysine 11 and lysine 18 via a conventional stepwise method. METHODS: The PEG-lipid was obtained by condensing palmitic acid derivative of epsilon-maleimido lysine with methoxy poly(ethylene glycol) amine. Under reductive conditions, the PEG-lipid readily reacted with sCT to yield the resultant compound, Mal-PL-sCT. KEY FINDINGS: Dynamic light scattering analyses suggested that Mal-PL-sCT and 2PEG-Mal-sCT exhibited robust helical structures with a high tendency to aggregate in water. Both compounds were more stable against intestinal degradation than sCT, although Mal-PL-sCT was less stable than 2PEG-Mal-sCT. However, 2PEG-Mal-sCT did not possess hypocalcaemic activity while Mal-PL-sCT retained the hypocalcaemic activity of sCT when it was subcutaneously injected in the rat model. Multiple functional groups may be conjugated to a peptide via a tri-block linker without the risk of obliterating the intrinsic bioactivity of the peptide. CONCLUSIONS: The resultant novel PEG-lipid has a potential role to optimize protein and peptide delivery.

Lipeo-sCT: a novel reversible lipidized salmon calcitonin derivative, its biophysical properties and hypocalcemic activity.

We have previously described the design and synthesis of Mal-sCT and compared its biological activity with its reversible counterpart, REAL-sCT. Mal-sCT was salmon calcitonin (sCT) conjugated with two molecules of an epsilon-maleimido lysine derivative of palmitic acid via non-reversible thioether bonds at its cysteine residues while REAL-sCT was sCT conjugated with two molecules of a cysteine derivative of palmitic acid via reducible disulfide bonds at its cysteine residues. Neither compounds when dissolved in water could reproducibly improved the oral deliverability of sCT. The purpose of this study was to characterize and evaluate Lipeo-sCT, a novel sCT analog conjugated via reducible disulfide bonds with two amphiphilic groups consisting of a hydrophobic hexadecyl moiety attached via an ether bond to a hydrophilic triethylene glycol moiety. Lipeo-sCT was successfully synthesized by a 4-step reaction, purified and identified by ESI-MS. Analysis by dynamic light scattering (DLS) and transmission electron microscopy (TEM) suggested it had a propensity to form aggregates in water, although the aggregation behavior was controllable by modulating solvent polarity. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay indicated a lack of cytotoxicity against the Caco-2 cells at up to 100 microM. Compared with sCT, Lipeo-sCT lowered plasma calcium to comparable levels when injected subcutaneously at 0.15 mg/kg into female Wistar rats, but the hypocalcemic activity of Lipeo-sCT was prolonged by at least 6 more hours. This was attributable to a continual regeneration of sCT from Lipeo-sCT. sCT was detectable in plasma 8h following subcutaneous injection of Lipeo-sCT (1.90 mg/kg), while Lipeo-sCT was not observed in plasma at all time points. By comparison, sCT was detectable in plasma for less than 2.5h following subcutaneous injection at an equivalent dose (1.50mg/kg). Data from this study complement those of previous studies, and add to the body of knowledge correlating the in vivo activity of lipidized peptides to their physical properties.

Synthesis, characterization and in vivo activity of salmon calcitonin coconjugated with lipid and polyethylene glycol.

An irreversible lipidized salmon calcitonin (sCT) analog, Mal-sCT, was previously shown to have comparable hypocalcemic activity to sCT in vivo. This study reports on the synthesis, characterization and pharmacological activity of novel PEGylated Mal-sCT analogs. Mal-sCT, prepared by conjugating sCT via thio-ether bonds with aqueous-soluble palmitic acid derivative at Cys 1 and Cys 7, was reacted with mPEG-succinimide (mPEG-Suc, 5 kDa). The products were purified and then identified by MALDI-TOF MS and HPLC. Mal-sCT was conjugated with 1 (1PEG-Mal-sCT) or 2 (2PEG-Mal-sCT) PEG chains at Lys 11 and Lys 18, the former being the preferred site of conjugation at higher mPEG-Suc/Mal-sCT ratio. Circular dichroism analysis showed the PEGylated Mal-sCT analogs to possess a robust helical conformation, while size measurement by dynamic light scattering indicated a propensity of the peptides to self-aggregate in aqueous solutions. Both 1PEG-Mal-sCT and 2PEG-Mal-sCT were more stable in rodent intestinal fluids than sCT or Mal-sCT. However, 1PEG-Mal-sCT had comparable hypocalcemic activity to Mal-sCT when injected subcutaneously in the rat, while 2PEG-Mal-sCT was inactive. 1PEG-Mal-sCT was inactive when administered orally in the rat. This study suggested PEGylation of Mal-sCT increased the stability of the lipidized peptide to enzyme degradation, but did not enhance its hypocalcemic activity.

Comparison of reversible and nonreversible aqueous-soluble lipidized conjugates of salmon calcitonin.

Reversible aqueous lipidization (REAL) at the interdisulfide bond has been shown to improve the deliverability of some peptide drugs. Recently, we developed a nonreversible aqueous lipidization method targeted at the interdisulfide bond of salmon calcitonin (sCT). The resultant derivative had comparable hypocalcemic activity to sCT after subcutaneous injection in rats, despite possessing significantly different biophysical properties. The purpose of this study was to conduct a comparative evaluation of the biophysical properties of the reversible aqueous-soluble lipidized sCT (REAL-sCT) and its corresponding nonreversible aqueous-soluble compound (Mal-sCT) with a view to correlate these properties to the bioactivities of the peptides. REAL-sCT and Mal-sCT were successfully synthesized, purified and identified. Both conjugates showed comparable retention times in a C-18 HPLC column, as well as robust helical structures and aggregation behavior in water, although REAL-sCT was shown by dynamic light scattering experiments to form larger aggregates than Mal-sCT in water. The larger particle size of REAL-sCT correlated with its stronger resistance to degradation by intestinal enzymes. Unlike Mal-sCT, REAL-sCT was rapidly converted to sCT in liver juice; however, the regenerated sCT appeared to degrade at a slower rate than unmodified sCT in the liver juice. Compared with sCT, REAL-sCT after subcutaneous injection as an aqueous solution at a dose of 0.15 mg/kg produced a prolonged hypocalcemic activity that lasted at least 24 h in the rat. Using a novel LC-MS/MS method that was developed for this study, we were able to show concomitant increases in REAL-sCT and sCT plasma concentrations with time, the latter prevailing at 10% the molar concentration of the former. In contrast, sCT was not present in the plasma following the subcutaneous injection of Mal-sCT, although a comparable hypocalcemic activity with shorter duration was observed. Oral administration of REAL-sCT and Mal-sCT as aqueous solutions at sCT equivalent dose of 5.0 mg/kg did not produce significant hypocalcemic activity. This study is the first thorough examination of the biophysical characteristics of the corresponding reversible and nonreversible aqueous-soluble lipidized peptide molecules. The results obtained should be useful for the development of the oral formulation of peptide and protein drugs.

Aqueous-soluble, non-reversible lipid conjugate of salmon calcitonin: synthesis, characterization and in vivo activity.

PURPOSE: A novel, non-reversible, aqueous-based lipidization strategy with palmitic acid as a model lipid was evaluated for conjugation with salmon calcitonin (sCT). MATERIALS AND METHODS: A water-soluble epsilon-maleimido lysine derivative of palmitic acid was synthesized from reaction of palmitic acid N-succinimidyl ester and epsilon-maleimido lysine. The latter was generated from reaction of alpha-Boc-lysine and methylpyrrolecarboxylate, with subsequent deprotection of the Boc group. The palmitic derivative was further conjugated with sCT via a thio-ether bond to produce Mal-sCT in aqueous solution. The identity and purity of Mal-sCT was confirmed by Electrospray Ionisation Mass spectrometry (ESI-MS) and HPLC. RESULTS: Yield of Mal-sCT was 83%. Dynamic light scattering and circular dichroism data suggested that Mal-sCT presented as a stable helical structure in aqueous solutions of varying polarity, with a propensity to aggregate at concentrations above 11 microM. Cellular uptake of Mal-sCT was twice that of sCT in the Caco-2 cell model, and the conjugate was more resistant to liver enzyme degradation. Mal-sCT exhibited comparable hypocalcemic activity to sCT when administered subcutaneously in the rat model at sCT equivalent dose of 0.114 mg/kg. Peroral Mal-sCT, however, produced variability in therapeutic outcome. While four out of six rats did not respond following intragastric gavage with Mal-sCT, two rats showed significantly suppressed plasma calcium levels (approximately 60% of baseline) for up to 10 h. CONCLUSION: A novel non-reversible, water-soluble lipid conjugate of sCT was successfully synthesized that showed (1) different aggregation behavior and secondary structure, (2) improved enzymatic stability and cellular uptake, and (3) comparable hypocalcemic activity in vivo compared to sCT.