Perfluorocarbon imaging in vivo: a 19F MRI study in tumor-bearing mice

Multiresonance perfluorocarbon emulsions (Oxypherol and Fluosol-DA) were imaged in tumor-bearing mice using 19F spin-echo magnetic resonance imaging in vivo. Multiple thin-slice fluorine images free of chemical shift artifacts were obtained in 13 minutes and these were correlated with proton images obtained during the same experiment to delineate the anatomic distribution of perfluorocarbons. Sequential images were used to determine the time course of the distribution and the retention of the compounds in tumors and organs. 19F MR spectroscopy was used ex vivo to determine with high sensitivity the relative concentration of perfluorocarbons in different tissues and organs and to confirm the results obtained from imaging experiments. The fluorine images visually demonstrated the preferential localization of the perfluorocarbons in the liver and spleen; shortly after injection, the images also revealed the highly vascularized tumor-chest wall interface. Imaging and spectroscopy together showed that the perfluorocarbons were removed from the blood pool within hours and remained sequestered in tissues at later times; the highest concentrations were found in the spleen and liver, where the agents were retained without spectral changes for the duration of these studies. The perfluorocarbons accumulated within tumors at dose-dependent concentrations, one to two orders of magnitude smaller than those observed in the spleen and liver.     

Intercompany Study to Evaluate the Robustness of Capillary Isoelectric Focusing Technology for the Analysis of Monoclonal Antibodies

Interlaboratory comparisons are essential to bringing emerging technologies into biopharmaceutical industry practice and regulatory acceptance. As a result, an international team including 12 laboratories from 10 independent biopharmaceutical companies in the United States and Switzerland was formed to evaluate the precision and robustness of capillary isoelectric focusing (CIEF) to assess the charge heterogeneity of monoclonal antibodies. The different laboratories determined the apparent pI and the relative distribution of the charge isoforms of a representative monoclonal antibody (rMAb) sample using the same CIEF method. Statistical evaluation of the data was performed to determine within and between-laboratory consistencies and outlying information. The apparent pI data generated for each charge variant peak showed very good precision between laboratories with percentage of RSD values of ??0.5%. Similarly, the percentage of RSD for the rMAb charge variants percent peak area values are ??4.4% across different laboratories with different analysts using different lots of ampholytes and multiple instruments. Taken together, these results validate the appropriate use of CIEF in the biopharmaceutical industry in support of regulatory submissions.     

A novel editing technique for F MRI: Molecule-specific imaging

A novel technique is proposed to facilitate the selective imaging of specific molecules from a mixture. The application of the technique presented here demonstrates the ability to selectively produce ¹⁹F MR images of either trifluoroacetic acid or the perfluorocarbon emulsion Oxypherol-ET (perfluorotributylamine), when both molecules are present simultaneously. Selective detection is based on the presence of homonuclear J-modulation in one molecule and differential spin-spin relaxation time (T₂). Perfluorotributylamine, an A₃B₂ system, is subject to homonuclear J-modulation, which produces a null signal from the antiphase components of the triplet (A₃) when an echo time is used in a spin-echo image. At this echo time the second molecule, in this example trifluoroacetic acid, a non-coupled spin system, is selectively imaged. At longer echo times, e.g., TE = 1/J there is substantial recovery of the J-modulated signal, which may be solely observed due to T₂ decay of the trifluoroacetic acid signal. The method is demonstrated both using phantoms and in vivo.     

Improved MR imaging in extremely inhomogeneous radio-frequency fields

An improvement to the method for imaging with extremely inhomogeneous radio-frequency fields is presented which uses a pseudo-noise-modulated selective (PNMS) excitation prepulse to randomize the signal from outside the selected slice. This prepulse reduces the problems that arise from imperfect subtraction involved with the one-dimensional ISIS-type slice selection procedures. The results demonstrate two to three times improvement in the signal-to-noise ratio and reduction in the image artifacts when this prepulse is used.     

Columella footplate motion and the cochlear microphonic potential in the embryo and hatchling chicken

A piezoelectric (PZE) vibrator was used to mechanically drive the columella footplate and stimulate the cochlea of chicken embryos and hatchlings. Our objectives were to characterize the motion of the PZE driver and determine the relationship between columella footplate motion (displacement/ velocity) and the cochlear microphonic recorded from the recessus scala tympani (CMrst). At each frequency, displacement of the PZE driver probe tip was linearly related to the applied voltage over a wide range of attenuation levels (-60 to -20 dBre:50 Vp-p). The mean displacement across frequencies (100-4000 Hz) was 0.221+/-0.042 micromp-p for a constant applied voltage level of -20 dBre:50 Vp-p. Displacement was within 1.5 dB of the mean for this stimulus level at all frequencies except for 4000 Hz, where it was approximately 3 dB higher (p < 0.01). CMrst amplitudes in hatchlings were larger than amplitudes in embryos (p=0.003). For a given frequency, CM was linearly related to footplate displacement and velocity at both ages. The transform ratio of CMrst/A (CM amplitude/displacement) increased at approximately 6 dB/octave at frequencies between 100 and 1000 Hz in hatchlings suggesting that cochlear impedance (Zc) was resistive at these frequencies. In a large fraction of the embryos, Zc exhibited reactive behavior.     

The transient growth of ammonium chloride dendrites

We report measurements of the initial growth and subsequent transient response of dendritic crystals of ammonium chloride grown from supersaturated aqueous solution. Starting from a small, nearly spherical seed held in unstable equilibrium, we lower the temperature to initiate growth. The growth speed and tip radius approach the same steady state values independent of initial seed size. We then explore the response of the growing dendrite to changes in temperature. The crystal adjusts quickly and smoothly to the new growth conditions, maintaining an approximately constant value of vρ² throughout. Dissolving dendrites, on the other hand, are not characterized by the same value of vρ².     

A Series of Collaborations Between Various Pharmaceutical Companies and Regulatory Authorities Concerning the Analysis of Biomolecules Using Capillary Electrophoresis

An international project team (including members from US, Canada and UK) has been formed from a number of interested biopharmaceutical companies and regulatory authorities to conduct a cross-organisation collaboration exercise. The results from this exercise demonstrate the robustness of CE-SDS across eight different organisations that used instruments of the same equipment model, the same reagents, and the same methodology. Data generated from the analysis of a series of molecular weight markers showed very good precision with regards to relative migration time (RMT) both within and between organisations. The apparent molecular weight of bovine serum albumin (BSA) was measured with good precision to within approximately 2% RSD across the participants. A representative IgG sample showed similar results with regards to relative migration time of its 3 main components, IgG Light Chain, IgG Non-glycosylated Heavy Chain, and IgG Heavy Chain. Fractional peak area for each peak also showed good agreement, with less than 9% RSD for all peaks. This exercise will facilitate both increased regulatory and industrial opinion of CE for biopharmaceutical analysis.CE in the Biotechnology & Pharmaceutical Industries: 7th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules, Montreal, Canada, August 12–16, 2005     

Energy conversion and high power pulse production using miniaturemagnetic flux compressors

The design, construction, and testing of miniature, high-power magnetic flux compressors are presented and discussed. The magnetic flux compressors are located inside high-speed, 30-mm projectiles that are launched with a high-pressure helium gun to velocities of approximately 300 m/s. The large deceleration force created when the projectile impacts the ground is used to power the magnetic flux compressor. Initial magnetic flux compressor currents of 200 mA are amplified up to 230 times to reach a peak current approaching 50 A. The output peak power is on the order of 3.5 kW into a 1-Ω load with a pulse length of 100 to 150 μs. Good agreement exists between the experimental results and the theoretical predictions for peak output current     

A Series of Collaborations between Various Pharmaceutical Companies and Regulatory Authorities Concerning the Analysis of Biomolecules Using Capillary Electrophoresis: Additional Instruments/Buffer

An international project team (including members from US, Canada and UK) was formed from a number of interested biopharmaceutical companies and regulatory authorities to conduct a cross-organisation collaboration exercise. The results of the first comparison with eight different organisations that used instruments of the same equipment model, the same reagents, and the same methodology has been reported previously [1]. This report represents the addition of other instruments using a different run buffer. The relative migration times were different, as expected, prohibiting a direct comparison between companies. The within-organisation variability was low for both relative migration time (<0.34% RSD% for all companies save one) and the peak area (<5% RSD% for all companies save one) when measuring the purity of a representative IgG sample. The apparent molecular weight of bovine serum albumin was measured with good precision (less than 10% RSD% across all companies) to the theoretical value when all data is utilized (67.5 kDa compared to 66.4 kDa). For a representative IgG sample, the three main components, IgG Light Chain, IgG Non-glycosylated Heavy Chain, and IgG Heavy Chain, could not be separated, specifically the IgG Non-glycosylated Heavy Chain and IgG Heavy Chain. When the IgG Non-glycosylated Heavy Chain and IgG Heavy Chain were combined for all organisations, the fractional peak area for the IgG Light Chain and IgG Non-glycosylated Heavy Chain + IgG Heavy Chain peak also showed excellent agreement, with less than 7.5 and 3.5% RSD%, respectively. The value of this exercise is in demonstrating the reliability of CE for the determination of apparent size of biopharmaceutical proteins. This underpins the appropriate use of such CE data in support of regulatory submissions.     

Flourine-19 NMR spectroscopy and imaging investigations of myocardial perfusion and cardiac function

Fluorine-19 nuclear magnetic resonance images of rabbit hearts have been obtained with the administration of a fluorinated contrast compound. The use of fluorine-labeled compounds allows differences in tissue perfusion to be demonstrated between hearts with and without coronary artery ligation. In postischemic reperfusion studies, regional hypoperfusion is also demonstrated by ¹⁹F imaging. The results of in vivo rabbit studies show that ¹⁹F NMR can be used to monitor the rate of blood pool and extravascular space dilution of a single bolus injection of either a water soluble or emulsified fluorine-labeled agent.