Kinetics and thermodynamics of dimer formation and dissociation for a recombinant humanized monoclonal antibody to vascular endothelial growth factor.

The recombinant humanized antibody (rhuMAb) VEGF has a high affinity for vascular endothelial growth factor and is currently being evaluated in clinical trials as a cancer therapeutic. Under acidic pH and low ionic strength conditions, the antibody was predominantly present as monomer. Under physiological conditions, the appearance of significant amounts of a noncovalent, reversible dimer were observed by size-exclusion chromatography. The kinetics and thermodynamics of the reversible self-association for rhuMAb VEGF monomer were investigated as a function of pH, temperature, and ionic strength by size-exclusion chromatography using the concentration jump method. The rate constant for dimer formation ranged 23-112 M(-)(1) min(-)(1) under the conditions studied, values that are significantly lower than those reported in the literature for other proteins that self-associate. The rate constant for dissociation ranged 0.0039-0.021 min(-)(1). Gibbs' free energies, enthalpies, entropies, and activation energies were determined and revealed that dimer formation is optimal at pH 7.5-8.0, which may be reflective of charge shielding occurring near the pI of the protein. There was a negative change in entropy for dissociation (values from -18.1 to -12.8 cal/mol K). In the presence of D(2)O or 1 M NaCl, dimerization was enhanced. The results of the kinetic and thermodynamic analysis of this study indicate that rhuMAb VEGF dimerization occurs primarily through hydrophobic interactions.

Lyophilization of protein formulations in vials: investigation of the relationship between resistance to vapor flow during primary drying and small-scale product collapse.

During the lyophilization process, formulations containing protein, bulking agent, or lyoprotectant form a dry product layer that can affect the transport of sublimed water vapor. We carried out an investigation of the primary drying segment of lyophilization to evaluate the relationship between the resistance to water vapor flow through the dried layer and the microstructure of the dried cake. Recombinant humanized antibody HER2 (rhuMAb HER2) formulated in trehalose was studied, as were protein-free formulations containing trehalose and sucrose. Sublimation rate and product temperature data were used to compute the resistance to mass transfer. Dried cake structure was examined by scanning electron microscopy and a novel fluorescence microscopy method. Collapse temperatures were determined by freeze-drying microscopy. Mass transfer resistance was found to decrease with increases in temperature for each material. Resistance also depended on composition, decreasing in the formulation series, rhuMAb HER2, trehalose, sucrose. The lyophilized material consisted of porous cakes, with a distinct denser region at the top. Formulation and temperature affected the microstructure of the dried cakes. The formulated trehalose and sucrose were seen, by both microscopy techniques, to possess small (2-20 microm) holes in their platelike structures after lyophilization. The quantity of holes was higher for material dried at higher temperature. The collapse temperature (Tc) of a material appeared to play a role in the process, as lower Tc was correlated with lower resistance and a greater extent of holes. Our results are consistent with the theory that lower resistance to water vapor flow in the primary drying stage of lyophilization may be due to small-scale product collapse.

The effect of benzyl alcohol on recombinant human interferon-gamma.

PURPOSE: The goal of this study was to investigate the conformational change and aggregation of recombinant human interferon-gamma (rhIFN-gamma) as a result of interaction between benzyl alcohol and the protein. The effects of buffer concentration, buffer species, ionic strength, rhIFN-gamma and benzyl alcohol concentrations on the dynamics of the interaction in liquid formulations were also examined. METHODS: The effect of benzyl alcohol on the secondary and tertiary structure of rhIFN-gamma in succinate and acetate buffers was studied using far-UV and near-UV circular dichroism spectrophotometry, respectively. Dynamic light scattering was employed to detect aggregate formation due to the interaction of benzyl alcohol with rhIFN-gamma. RESULTS: The addition of benzyl alcohol at 0.9% (w/v) in various liquid rhIFN-gamma formulations induced changes in circular dichroism (CD) spectra of the protein in the near-UV region, while the CD spectra in the far-UV region remained unaltered. There were gradual decreases in ellipticity with time throughout the near-UV CD spectra. The decreases in near-UV ellipticity induced by benzyl alcohol were accompanied by the formation of high molecular weight aggregates as measured by dynamic light scattering. Loss in near-UV ellipticity was accelerated at lower protein concentration and by increasing buffer or benzyl alcohol concentration. It was also faster in succinate than in acetate buffer. Formulation ionic strength did not affect the CD spectral changes in both the near- and far-UV regions. CONCLUSIONS: Interaction between benzyl alcohol and rhIFN-gamma is formulation dependent. Protein concentration, buffer species, buffer concentration, and preservative concentration play a significant role in determining the extent of the interaction and consequently the stability of the product.

Surface expression, polarization, and functional significance of CD73 in human intestinal epithelia.

During active intestinal inflammation polymorphonuclear leukocytes (PMN) transmigrate into the lumen and release 5'-AMP (J. Clin. Invest. 1993. 91:2320-2325). 5'-AMP is converted to adenosine by the apical epithelial surface with subsequent activation of electrogenic Cl- secretion (the basis of secretory diarrhea) via apical A2b adenosine receptors (J. Biol. Chem. 1995. 270:2387-2394). Using a polarized human intestinal epithelial monolayer (T84), we now characterize the basis of the observed conversion of 5'-AMP to adenosine required for this paracrine signaling pathway. An inhibitor of the ecto-5'-nucleotidase CD73, alpha, beta-methylene ADP (AOPCP), inhibited epithelial Cl- secretory responses to 5'-AMP, but not to authentic adenosine. Confocal immunofluorescent microscopy revealed CD73 to be surface expressed on both model and natural human intestinal epithelia. Expression was about sixfold greater on the apical cell surface as assessed biochemically by selective cell surface biotinylation, and morphologically by immunofluorescence. Treatment with phosphotidylinositol specific-phospholipase C (PI-PLC) released 95% of apical CD73, indicating that the intestinal CD73 possesses a glycosylphosphatidylinositol (GPI) anchor. Neither adenosine nor 5'-AMP stimulation induced intact T84 cells to shed surface CD73. The bulk of apical CD73 ( approximately 60%) was released from the cell surface by treatment with 1% Triton X-100 (TX-100) at 4 degrees C, but such release was not affected by pretreatment with ligand or by prior, antibody-mediated cross-linking of CD73. Subsequent analyses showed that the subpool of CD73 released by TX-100 at 4 degrees C was not truly solubilized, but rather represented TX-100-induced release of CD73-containing membrane fragments. These membrane fragments displayed light density on sucrose gradients characteristic of detergent insoluble glycosphingolipid-rich membrane domains (DIGs)/ caveolae, were solubilized by n-octyl glucoside (NOG, 1%) at 4 degrees C, and contained caveolin. These data indicate that human intestinal epithelia express CD73, which is apically polarized and targeted to microdomains with DIGs/caveolae characteristics. CD73 likely participates in translating paracrine, PMN-derived 5'-AMP signals to the authentic effector adenosine. These studies define CD73 as central to PMN-mediated intestinal Cl- secretion, the major directacting mechanism by which PMN induce intestinal epithelial Cl- secretion.

Inhibitory effect of sugars and polyols on the metal-catalyzed oxidation of human relaxin.

Previously, our laboratory (Li, S.; et al. Biochemistry 1995, 34, 5762-5772) showed that the oxidation of recombinant human relaxin (Rix) could be induced by ascorbic acid (AsA)/Cu(II), a system used for the metal-catalyzed generation of reactive oxygen species. In this study, we observed that this oxidation could be inhibited by high concentrations of mannitol and other sugars and polyols, such as ethylene glycol, glycerol, glucose, and dextran. Similar protective effects with high concentrations of mannitol were also observed in the AsA/CuCl2-induced oxidation of Gly-Met-Gly and Gly-His-Gly. In contrast, (carboxymethyl)cellulose had no protective effect on the metal-catalyzed oxidation of Rix. These results, together with results from deuterium isotope experiments and spectroscopic experiments, suggest that the inhibitory effect of polyols and sugars is probably due to the complexation of transition metal ions rather than a hydroxyl radical scavenging mechanism. However, dextran, a high molecular weight polysaccharide, might function as a hydroxyl radical scavenger to protect Rix from the metal-catalyzed oxidation.

Effects of reducing sugars on the chemical stability of human relaxin in the lyophilized state.

Sugars and polyols have been used routinely with lyophilized proteins and peptides as bulking agents, cryoprotectants, and lyoprotectants. However, reducing sugars may present a problem as excipients since they are potentially reactive with proteins. In this stability study of recombinant human relaxin (Rix) with various sugars as excipients in lyophilized formulations, we observed rapid covalent modifications of the protein in the presence of glucose. Analysis of the protein by LC/MS and tryptic mapping indicated two major degradation pathways. Covalent adducts of glucose with amino groups on the side chains of the protein (i.e., Lys and Arg) formed via the Maillard reaction. In addition, a significant amount of Ser cleavage from the C-terminal of the B-chain of relaxin was also identified when glucose was used as the excipient. It was observed that the latter reaction occurred to a greater extent in the solid state than in solution. We proposed a mechanism for this reaction involving an initial reaction of the Ser hydroxyl group with glucose followed by subsequent hydrolysis of the Trp-Ser amide bond via a cyclic intermediate. In contrast to glucose, mannitol (polyhydric alcohol) and trehalose (nonreducing sugar) produced stable, lyophilized formulations of Rix.

Compaction assay: a rapid and simple in vitro method to assess the responsiveness of a biopolymer matrix to enzymatic modification.

A rapid and simple in vitro method is described which measures the extent of unrecoiled solids compression when a complex biopolymer is subjected to a centrifugal force. This method, termed the compaction assay, was used to assess the response of purulent cystic fibrosis (CF) sputum samples to the addition of recombinant human deoxyribonuclease I (rhDNase). Enzyme treatment resulted in a dramatic decrease in DNA size, a redistribution of total DNA content from the pellet to supernatant, a significant decrease in that pellet volume and a decrease in elastic modulus. Sample elasticity, measured by a dynamic cone and plate viscometer, could be related to compaction assay results. These results suggest that the compaction assay may be a useful in vitro method for rapidly assessing the actions of enzymatic disruption of a complex biopolymer, such as that observed for the actions of rhDNase on purulent airway secretions.

Addition of a bacterial alginate lyase to purulent CF sputum in vitro can result in the disruption of alginate and modification of sputum viscoelasticity.

Alginate is a large molecular weight exopolysaccharide present in the purulent airway secretions of cystic fibrosis (CF) patients. This polymer, produced by some of the opportunistic pathogens associated with the recurrent lung infections characteristic of CF, has been suggested to effect an increase in the viscoelastic properties of purulent CF airway secretions. We have investigated the use of an enzyme targeted at this exopolysaccharide, an alginate lyase obtained from a bacterial source, to disrupt its polymeric nature and effect a change in the rheological properties of CF sputum in vitro. Expectorated sputum samples obtained from hospitalized CF patients were found to contain 80-200 micrograms alginate per ml sputum with no measurable endogenous alginate lyase activity. Treatment with exogenous alginate lyase prepared from a mucoid strain of Pseudomonas aeruginosa resulted in the disruption of alginate and a decrease in sputum viscoelasticity in a small percentage of the samples tested. Similar treatment of these samples with recombinant human deoxyribonuclease I to cleave DNA present in purulent sputum and the use of alginate extracted from sputum as an alginate lyase assay substrate suggested that the inability of the exogenous alginate lyase to disrupt sputum alginate was not due to substrate inaccessibility or an unresponsive substrate. Concentrations of Ca2+ and Zn2+ in alginate lyase-resistant sputum samples, determined by metal ion analysis, were found to inhibit enzyme activity in studies using seaweed alginate as a substrate. High concentrations of Ca2+ and Zn2+ in sputum samples initially resistant to lyase activity could be reduced significantly in some samples by dialysis and these same samples acquired sensitivity to the lyase. Other sputum samples did not show reduced concentrations of Ca2+ and Zn2+ following dialysis and these samples remained lyase-insensitive. Together, these results suggest that bacterial alginate present within purulent CF sputum may be quite stable, that endogenous alginate lyase activities appear to be limited and that the in vitro addition of exogenous alginate lyase can lead to the disruption of alginate and a change in the viscoelastic properties of some purulent CF sputum samples.

Activated eosinophils evoke chloride secretion in model intestinal epithelia primarily via regulated release of 5'-AMP.

Eosinophils may be prominent in intestinal diseases including allergic gastroenteritis, inflammatory bowel disease, enteritis associated with hypereosinophilic syndromes (HES), and parasitic diseases. Unlike normal blood eosinophils, those that circulate in HES and those that infiltrate inflamed tissue exhibit an "activated" phenotype. To model intestinal epithelial-eosinophil interactions, we used peripheral blood eosinophils and human crypt-like T84 epithelial cell-line monolayers. Eosinophils from normal, mildly atopic donors, only if activated by PMA or primed with granulocyte-macrophage-CSF for 48 h, as well as eosinophils from HES patients elicited a short circuit current when applied apically to T84 monolayers. This eosinophil-derived bioactivity, which was transferable in cell-free supernatants and in < 1000 m.w. ultrafiltrates, stimulated electrogenic Cl- secretion, as indicated by inhibition with basolateral bumetanide or gluconate substitution and by enhancement of the rate constant for 125I efflux from preloaded T84 cells. This secretagogue activity was blocked in both intact activated eosinophils and in eosinophil-conditioned supernatants, by 8-phenyl-theophylline, indicating involvement of an adenosine receptor. Ion exchange and reversed-phase HPLC analyses demonstrated that eosinophil supernatant ultrafiltrates contained elevated levels of 5'-AMP that was converted to adenosine after incubation with epithelium. Inhibition of epithelial apical membrane ecto-5'-nucleotidase ablated the conversion to adenosine. These studies establish that activated eosinophils elicit Cl- secretion from intestinal epithelial and that 5'-AMP released by eosinophils followed by its conversion to adenosine at the epithelial surface is the basis for this response.

The application of size exclusion chromatography and computer simulation to study the thermodynamic and kinetic parameters for short-lived dissociable protein aggregates.

We describe a method to study the monomer-dimer equilibrium of human growth hormone (hGH) making use of a very short size exclusion high-performance liquid chromatographic column and rapid flow rates. By adjusting the flow rate and thus the retention time on the column, the dissociation of the hGH dimer can be observed. Using computer simulation, both the equilibrium constant for dissociation and the dissociation rate constant can be determined directly, followed by the indirect determination of the association rate constant. This method is potentially useful for determining the thermodynamic the kinetic parameters of aggregation of many protein-protein (homodimers and heterodimers) and protein-ligand systems whose rates of interaction are too rapid to be studied by conventional techniques.

5'-adenosine monophosphate is the neutrophil-derived paracrine factor that elicits chloride secretion from T84 intestinal epithelial cell monolayers.

Neutrophil transmigration across intestinal epithelia is thought to contribute to epithelial dysfunction and characterizes many inflammatory intestinal diseases. Neutrophils activated by factors, normally present in the lumen, release a neutrophil-derived secretagogue activity to which intestinal epithelia respond with an electrogenic chloride secretion, the transport event which underlies secretory diarrhea. Using sequential ultrafiltration, column chromatographic, and mass and Raman spectroscopic techniques, neutrophil-derived secretagogue was identified as 5'-AMP. Additional studies suggested that neutrophil-derived 5'-AMP is subsequently converted to adenosine at the epithelial cell surface by ecto-5'-nucleotidase and that adenosine subsequently activates intestinal secretion through adenosine receptors on the apical membrane of target intestinal epithelial cells. These findings suggest that this ATP metabolite may serve as a neutrophil-derived paracrine mediator that contributes to secretory diarrhea in states of intestinal inflammation.

Intranasal bioavailability of insulin powder formulations: effect of permeation enhancer-to-protein ratio.

The intranasal administration of powder formulations containing insulin and the permeation enhancer sodium tauro-24,25-dihydrofusidate (STDHF) were investigated in the sheep model. Both the hypoglycemic response and the serum insulin levels increased as the mole ratio of STDHF to insulin was increased from 0 to 16.8. In vitro dissolution rates of the powders and the rapid tmax (approximately 5 min) observed after intranasal administration suggest that the absorption of insulin is not dissolution limited. The bioavailabilities (F) of the powder formulations ranged from 2.9 to 37.8%. In comparison, the F values for a solution formulation with a STDHF:insulin ratio of 8.4 administered as either drops or spray were 15.7 and 37.4%, respectively. The permeation enhancer STDHF increases mucosal permeability and reduces the average molecular weight of the insulin species.

Multiple equilibria binding treatment of lipid and detergent interactions with membrane proteins. Application to cytochrome c oxidase solubilized in cholate.

A modified multiple binding equilibria treatment is presented that allows determination of thermodynamic parameters of the interaction of phospholipids with integral membrane proteins solubilized in excess detergent. Lipid binding is modeled as a series of exchange reactions between lipid molecules and detergent molecules at the hydrophobic protein surface. A general equation is derived which expresses a relative association constant (K) and the total number of contact sites at the lipid-protein interface (N) in terms of experimentally measurable variables. A useful simplification of the general equation occurs when the amount of detergent is high relative to the total number of lipid binding sites in the sample. Computer simulations show that in cases we have examined there appears to be an experimentally accessible range of detergent to protein molar ratios where the approximation at high detergent is useful for analyzing experimental data. This model is used to examine the competition between cholate and spin-labeled phospholipids for the hydrophobic surfaces of bovine heart cytochrome c oxidase. We find, for example, that K = 12 +/- 2 for phosphatidylcholine relative to cholate (i.e., the cholate molecules are relatively easily displaced by membrane lipids). This helps to explain the experimental observation that cholate is an effective detergent both for solubilizing cytochrome c oxidase and for reconstituting this protein into a defined lipid bilayer environment. An excess of cholate readily displaces almost all of the native phospholipids, and the protein is dispersed in cholate micelles. However, when phospholipids are added back, the cholate molecules at the protein surface are replaced because of the higher relative binding of the phospholipids. Observed differences between the behavior of phosphatidylcholine and phosphatidylglycerol suggest that reconstitution in cholate is a selective process in which detergent molecules in localized areas on the protein surface are more readily displaced by certain phospholipids.

Raman spectra of the model B-DNA oligomer d(CGCGAATTCGCG)2 and of the DNA in living salmon sperm show that both have very similar B-type conformations.

Raman spectra were obtained from aqueous solutions of the deoxyoligonucleotide d(CGCGAATTCGCG)2 (I), which has been suggested as a model for B-type DNA conformation. These spectra were compared with the Raman spectra of the aqueous solutions of several DNAs of natural origin taken under identical solution conditions. Since the model sequence has a high percent GC (66%), the Raman spectrum was compared with the Raman spectrum of the DNA from Micrococcus lysodeikticus (72% GC), and the spectra of the two different DNAs were found to be rather similar in both 50 mM salt and 6 M salt solutions. Computer-aided band-shape analysis of the backbone vibrational region of the Raman spectra shows the existence of several bands corresponding to different furanose ring puckers. This appears to indicate a heterogeneity of furanose ring pucker in both the model dodecamer and the native DNA. Significant differences were found in the intensity of the conformational marker band at 810 cm-1, which indicates corresponding differences in furanose ring pucker heterogeneities in these two high GC content DNAs. The Raman spectrum of the dodecamer (I) was used to analyze the Raman spectrum of the DNA inside the head of living intact salmon sperm. Sperm spectra were taken with both our conventional Raman spectrograph and a newly developed intracavity laser Raman microscope system. Although the DNA in the sperm head is required by packing considerations to be in a highly compact and condensed state, the Raman spectra of the intact sperm are almost identical with that of the model dodecamer (I) if the difference in base composition is taken into account.(ABSTRACT TRUNCATED AT 250 WORDS)