Highly concentrated monoclonal antibody solutions: direct analysis of physical structure and thermal stability.

Virtually all current analytical methods employed in the development of highly concentrated monoclonal antibody (MAb) formulations require dilution of the sample before acquiring data. Thus, there is an unmet need for methods to study proteins directly at high concentration, since extrapolation of stability indicating parameters obtained from dilute studies may not be representative of the high concentration solution. Only slight or no modifications of biophysical methods including fluorescence, UV absorbance, circular dichroism, and FTIR (ATR and transmittance) spectroscopies as well as differential scanning calorimetry (DSC) are described here that permit the direct study of immunoglobulins (and other proteins) at high concentrations. Although FTIR spectra show differences that are dependent upon sampling geometry, other spectroscopic data from two different recombinant MAbs suggests that structure of each antibody exists in a physically similar state in the concentration range of 0.1-190 mg/mL in 40 mM pH 6 citrate-phosphate buffer. Upon thermally stressing these proteins, spectroscopic techniques that probe tertiary structure demonstrate a decrease in the apparent thermal melting temperature of approximately 5-20 degrees C of both proteins with increasing concentration. In contrast, DSC thermograms and CD thermal experiments suggest a minor degree of stabilization (approximately 2 degrees C) for both antibodies although protein association could be responsible for these observations. Empirical phase diagrams produced from spectroscopic data also suggest (1) the existence of similar structural states at low temperatures independent of concentration and (2) a decrease in the temperature at which phase changes are observed with increasing concentration. The decrease in structural stability observed in these studies is probably the result of aggregation or self-association of the recombinant MAbs upon heating in crowded solutions and not due to a decrease in the intrinsic structural stability of the MAbs.

Synthesis and anticonvulsant activity of 3-aryl-5H-2,3-benzodiazephine AMPA antagonists.

A novel series of 3-aryl-5H-2,3-benzodiazepines with N-3 aromatic substituents has been synthesized. Good in vivo anticonvulsant activity of the new compounds has been demonstrated employing the maximal electroshock seizure test in mice. Evaluation of a subset of the compounds in the cortical wedge assay confirmed the new structures to be AMPA antagonists.

Problems associated with use of the benzyloxymethyl protecting group for histidines. Formaldehyde adducts formed during cleavage by hydrogen fluoride.

The use of N alpha-tert.-butyloxycarbonyl-N pi-benzyloxymethylhistidine in peptide synthesis resulted in significant levels of several different side products attributable to the generation of formaldehyde during the hydrogen fluoride cleavage reaction. Methylated impurities in a decapeptide were isolated and identified. These methylated impurities were attributed to the use of the benzyloxymethyl protecting group for the histidines, since the impurities did not form when the dinitrophenyl protecting group was used. Also, peptides containing benzyloxymethyl-protected histidines in addition to N-terminal cysteines quantitatively yielded their respective N-terminal thiazolidine derivatives upon isolation from standard hydrogen fluoride cleavage mixtures. Thiazolidine ring formation was circumvented by including in the cleavage reaction a formaldehyde scavenger such as cysteine hydrochloride or resorcinol.

Mapping an antibody-binding site and a T-cell-stimulating site on the 1A protein of respiratory syncytial virus.

A synthetic peptide modeled on residues 45 to 60 of the 1A protein of respiratory syncytial (RS) virus [1A(45-60)] was constructed and used for immunization of mice and rabbits. The immunoglobulin G fraction of the resulting rabbit antibody, purified on protein A-Sepharose, immunoprecipitated from RS-infected HEp-2 cells a protein with a molecular size of approximately 9.5 kilodaltons, which corresponds to the previously published molecular size of the 1A protein (Y. T. Huang, P. L. Collins, and G. W. Wertz, Virus Res. 2:157-173, 1985). To investigate the T-cell-inducing properties of 1A(45-60), six strains of mice were immunized and their popliteal lymph node cells were tested for proliferation upon restimulation with peptide in vitro. The lymph node cells of all six strains of mice were responsive to restimulation with 1A(45-60) and showed high- and low-responder strain variation. These peptide-primed lymph node cells also proliferated upon in vitro restimulation with RS virus-infected cells. Correlation of proliferation with interleukin 2 production suggested that the responding lymphocytes were T-helper cells. The antibody-binding and T-cell-stimulating sites of 1A were mapped by constructing a series of overlapping synthetic peptides and testing each for ability to react with antiserum prepared by immunization of BALB/C mice with free peptide 1A(45-60) or for ability to restimulate proliferation in 1A(45-60)-primed lymph node cells of BALB/C mice. Human antibody, obtained during confirmed RS virus infection, was similarly tested with the truncated peptides. Antibody-binding activity was reduced after truncation from the carboxy terminus, and a binding site was mapped to residues 51 through 60, the smallest peptide tested. T-cell-stimulating activity in mice was relatively resistant to truncation from the carboxy terminus and sensitive to truncation from the amino terminus. The smallest region which retained significant T-cell-stimulating activity mapped to residues 46 through 56. However, addition of the naturally occurring Cys at residue 45 and extension of the C terminus to residue 62 resulted in maximum T-cell-stimulating activity of the peptide. These data define both a T-cell epitope and a B-cell epitope of the 1A protein of RS virus and suggest that the carboxy terminus of 1A contains a B-cell epitope, involving residues 51 through 60, which is recognized during natural human infection.