Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometry

Recombinant monoclonal antibody drug products play an increasingly important role in the treatment of various diseases. Antibodies are large, multi-chain proteins and antibody preparations often contain several molecular variants, which renders them heterogeneous. The heterogeneity is further increased in immunoconjugates prepared by covalently linking several drug molecules per antibody molecule. As part of the product characterization, the molecular weights of the antibodies or their drug conjugates need to be measured. Electrospray ionization mass spectrometry (ESI-MS) is well suited for the analysis of recombinant antibodies and immunoconjugates. Sample preparation is an important element of ESI-MS analysis, in particular samples need to be freed of interfering charged species, such as salts and buffer components. In this paper, Amicon centrifugal filters, reversed-phase high-performance liquid chromatography (HPLC), and size-exclusion HPLC were evaluated for sample desalting. Size-exclusion HPLC, using aqueous acetonitrile as the mobile phase, directly coupled to ESI-MS provided the best performance and was optimized for the study of immunoconjugates. The results showed that antibodies carrying covalently linked maytansinoid molecules generated charge envelope profiles that differ from those of the non-conjugated antibody. For the determination of the distribution of the various conjugate species in an immunoconjugate sample prepared by randomly linking in the average 3.6 drug molecules per antibody molecule, the experimental conditions needed to be carefully selected to allow acquisition of the whole spectrum containing the charge envelopes of all species.     

Bilayer vesicles of amphiphilic cyclodextrins: host membranes that recognize guest molecules

A family of amphiphilic cyclodextrins (6, 7) has been prepared through 6-S-alkylation (alkyl=n-dodecyl and n-hexadecyl) of the primary side and 2-O-PEGylation of the secondary side of alpha-, beta-, and gamma-cyclodextrins (PEG=poly(ethylene glycol)). These cyclodextrins form nonionic bilayer vesicles in aqueous solution. The bilayer vesicles were characterized by transmission electron microscopy, dynamic light scattering, dye encapsulation, and capillary electrophoresis. The molecular packing of the amphiphilic cyclodextrins was investigated by using small-angle X-ray diffraction of bilayers deposited on glass and pressure-area isotherms obtained from Langmuir monolayers on the air-water interface. The bilayer thickness is dependent on the chain length, whereas the average molecular surface area scales with the cyclodextrin ring size. The alkyl chains of the cyclodextrins in the bilayer are deeply interdigitated. Molecular recognition of a hydrophobic anion (adamantane carboxylate) by the cyclodextrin vesicles was investigated by using capillary electrophoresis, thereby exploiting the increase in electrophoretic mobility that occurs when the hydrophobic anions bind to the nonionic cyclodextrin vesicles. It was found that in spite of the presence of oligo(ethylene glycol) substituents, the beta-cyclodextrin vesicles retain their characteristic affinity for adamantane carboxylate (association constant K(a)=7.1 x 10(3) M(-1)), whereas gamma-cyclodextrin vesicles have less affinity (K(a)=3.2 x 10(3) M(-1)), and alpha-cyclodextrin or non-cyclodextrin, nonionic vesicles have very little affinity (K(a) approximately 100 M(-1)). Specific binding of the adamantane carboxylate to beta-cyclodextrin vesicles was also evident in competition experiments with beta-cyclodextrin in solution. Hence, the cyclodextrin vesicles can function as host bilayer membranes that recognize small guest molecules by specific noncovalent interaction.     

Spectrofluorometric method for determination of the total mercury content in environmental samples — Waste waters

A highly selective spectrofluorometric method for the determination of total mercury (Hg) in waste waters is described. Fluorescence quenching of rhodamine B with Hg(II) in the presence of iodide, after a concentration step, is the basis of this sensitive method. All forms of mercury, including organic compounds, are pre-oxidized to ionic mercury by acidic potassium permanganate. The final and complete oxidation is achieved by adding potassium persulphate and heating. Hg(II) was reduced by tin(II) chloride and Hg vapour driven by an air stream into an absorption solution containing potassium permanganate and sulphuric acid, using a closed, recirculating air stream. In this solution fluorescence quenching of rhodamine B at an excitation wavelength of 485 nm and emission wavelength of 586 nm was measured. The recoveries were done by adding 3.0 g Hg/100 ml to each sample before the digestion. It was indicated that the recoveries for determining mercury in waste waters were 98.3%–102.7%. The method gives reliable results down to a concentration of 10 ng Hg/ml waste water.     

Subtractive differential pulse anodic stripping voltammetry at a stationary mercury-coated glassy carbon electrode

The application of subtractive mode differential pulse anodic stripping voltammetry (SDPASV) at a stationary mercury-coated glassy carbon electrode for the analysis of labile Zn(II), Cd(II), Pb(II) and Cu(II) is described. It is shown that the method has an improved sensitivity to Cu(II) owing to elimination of high background currents normally encountered in normal mode DPASV at the TFME. The sensitivity limits of the present method to Cd(II) and Cu(II) is estimated to be 0.025 and 0.067 ppb respectively, when a 2 min deposition time is used. It is suggested that the high sensitivity of the method coupled to the relative simplicity of the stationary electrodes could make the method useful in environmental and natural water studies.     

On banach spaces whose duals areL 1 spaces

A structure theorem for Banach spaces whose duals areL ₁ spaces, is proved. The research of the second named author has been sponsored by the Air Force Office of Scientific Research under Grant AF EOAR 66-18 through the European Office of Aerospace Research (OAR) United States Air Force.     

The motion of incompressible fluids with electrical resistivity past thin airfoils in oblique fields

Résumé Le problème de l'écoulement des fluides à résistivité électrique en présence d'un profil mince, dans le cas des champs obliques, est réduit à la résolution d'une équation intégrale du type Fredholm. L'équation peut être résolue à l'aide de la méthode des approximations successives. Dans le cas des champs alignés et dans celui des champs perpendiculaires, lorsque pourR (nombre magnétique de Reynolds) nous considérons des valeurs petites, les équations se résolvent de façon explicite et exacte. On détermine la portance dans le cas d'une plaque et on montre la manière dont la portance décroit à mesure queR augmente. On montre également que dans le cas des champs orthogonaux, la portance est plus petite que dans celui des champs alignés. De la solution générale on obtient, comme cas particulier, la solution de l'aérodynamique classique ainsi que la solution du cas du fluide à résistivité négligeable.     

Mineral?le

Ohne Zusammenfassung