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Biomedical subjects

M J Hageman

Publications and source records attributed to M J Hageman.

At least 19 recordsLinked to original sources
PubMed2002

Effect of viscosity on the deamidation rate of a model Asn-hexapeptide.

The effect of viscosity on the deamidation rate of a model Asn-containing hexapeptide (l-Val-l-Tyr-Pro-l-Asn-Gly-l-Ala) was assessed in aqueous solution and in solids containing varying amounts of poly(vinyl pyrrolidone) (PVP) and water. Stability studies were conducted at 0.1 mg/mL peptide and 0-50% PVP (w/w) in aqueous solution, and at 5% (w/w) peptide and different relative humidities (31.6, 53.1, 74.4 and 96%) in the solid state. The parent peptide and its deamidation products were analysed by reverse-phase high-performance liquid chromatography. Deamidation rates decreased with increasing solvent viscosity in a manner described by a semi-empirical mathematical model developed to describe this relationship. The results suggest that the motion of the Asn side-chain along the reaction coordinate is a function of the macroscopic solvent viscosity. However, the apparent energy barrier for the diffusive movement of the side-chain appears to be less than the energy barrier for that associated with macroscopic viscosity. The dependence of the deamidation rate on viscosity in both viscous solution and hydrated solids further demonstrates the importance of mobility in peptide deamidation.

Calorimetry, Differential Scanning↗
PubMed1999

Chemical stability of peptides in polymers. 1. Effect of water on peptide deamidation in poly(vinyl alcohol) and poly(vinyl pyrrolidone) matrixes.

This paper examines the effect of water content, water activity, and glass transition temperature (T(g)) on the deamidation of an asparagine-containing hexapeptide (VYPNGA; Asn-hexapeptide) in lyophilized poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) at 50 degrees C. The rate of Asn-hexapeptide deamidation increases with increasing water content or water activity and, hence, decreasing T(g). The rate of deamidation is more sensitive to changes in these parameters in PVA than in PVP. Deamidation is clearly evident in the glassy state in both formulations. In the glassy state, the peptide is more stable in PVA than in PVP formulations but is less stable in the rubbery state. No single variable (water content, water activity, or T(g)) could account for the variation in deamidation rates in PVA and PVP formulations. Deamidation rates were correlated with the degree of plasticization by water (distance of T(g) from the dry intrinsic glass transition temperature); coincident curves for the two polymers were obtained with this correlation. Deamidation in PVA and PVP was closely correlated with the extent of water-induced plasticization experienced by the formulation relative to its glass transition at 50 degrees C, suggesting that the physical state of formulations could be used to predict chemical stability.

Adsorption↗
PubMed1999

Chemical stability of peptides in polymers. 2. Discriminating between solvent and plasticizing effects of water on peptide deamidation in poly(vinylpyrrolidone).

The mechanistic role of water in the deamidation of a model asparagine-containing hexapeptide (Val-Tyr-Pro-Asn-Gly-Ala) in lyophilized formulations containing poly(vinylpyrrolidone) (PVP) and glycerol was investigated. Glycerol was used as a plasticizer to vary formulation glass transition temperature (T(g)) without significantly changing water content or activity. Increases in moisture and glycerol contents increased the rate of peptide deamidation. This increase was strongly correlated with T(g) at constant water content and activity, suggesting that increased matrix mobility facilitates deamidation. In rubbery systems (T > T(g)), deamidation rates appeared to be independent of water content and activity in formulations with similar T(g)s. However, in glassy formulations with similar T(g)s, deamidation increased with water content, suggesting a solvent/medium effect of water on reactivity in this regime. An increase in water content also affected the degradation product distribution; less of the cyclic imide intermediate and more of the hydrolytic products, isoAsp- and Asp-hexapeptides, were observed as water content increased. Thus, residual water appears to facilitate deamidation in these solid PVP formulations both by enhancing molecular mobility and by solvent/medium effects, and also participates as a chemical reactant in the subsequent breakdown of the cyclic imide.

Adsorption↗
PubMed1999

Effects of buffer composition and processing conditions on aggregation of bovine IgG during freeze-drying.

The objective of this study was to identify critical formulation and processing variables affecting aggregation of bovine IgG during freeze-drying when no lyoprotective solute is used. Parameters examined were phosphate buffer concentration and counterion (Na versus K phosphate), added salts, cooling rate, IgG concentration, residual moisture level, and presence of a surfactant. No soluble aggregates were detected in any formulation after either freezing/thawing or freeze-drying. No insoluble aggregates were detected in any formulation after freezing, but insoluble aggregate levels were always detectable after freeze-drying. The data are consistent with a mechanism of aggregate formation involving denaturation of IgG at the ice/freeze-concentrate interface which is reversible upon freeze-thawing, but becomes irreversible after freeze-drying and reconstitution. Rapid cooling (by quenching in liquid nitrogen) results in more and larger aggregates than slow cooling on the shelf of the freeze-dryer. This observation is consistent with surface area measurements and environmental electron microscopic data showing a higher surface area of freeze-dried solids after fast cooling. Annealing of rapidly cooled solutions results in significantly less aggregation in reconstituted freeze-dried solids than in nonannealed controls, with a corresponding decrease in specific surface area of the freeze-dried, annealed system. Increasing the concentration of IgG significantly improves the stability of IgG against freeze-drying-induced aggregation, which may be explained by a smaller percentage of the protein residing at the ice/freeze-concentrate interface as IgG concentration is increased. A sodium phosphate buffer system consistently results in more turbid reconstituted solids than a potassium phosphate buffer system at the same concentration, but this effect is not attributable to a pH shift during freezing. Added salts such as NaCl or KCl contribute markedly to insoluble aggregate formation. Both sodium and potassium chloride contribute more to turbidity of the reconstituted solid than either sodium or potassium phosphate buffers at similar ionic strength, with sodium chloride resulting in a substantially higher level of aggregates than potassium chloride. At a given cooling rate, the specific surface area of dried solids is approximately a factor of 2 higher for the formulation containing sodium chloride than the formulation containing potassium chloride. Turbidity is also influenced by the extent of secondary drying, which underscores the importance of minimizing secondary drying of this system. Including a surfactant such as polysorbate 80, either in the formulation or in the water used for reconstitution, decreased, but did not eliminate, insoluble aggregates. There was no correlation between pharmaceutically acceptability of the freeze-dried cake and insoluble aggregate levels in the reconstituted product.

Animals↗
PubMed1997

Effects of bovine somatotropin (rbSt) concentration at different moisture levels on the physical stability of sucrose in freeze-dried rbSt/sucrose mixtures.

The inherent instability of many proteins during freeze-drying and storage necessitates the addition of excipients to protect the proteins. It is emphasized in the literature that lyophilized sugar/protein composites should be stored at temperatures below their glass transition temperature (T(g)) to prevent crystallization of excipients. The influence of bovine somatotropin (rbSt) concentration on inhibition of sucrose crystallization at different relative humidities (RH) was of interest. Thermally modulated differential scanning calorimetry (MDSC) was used to measure T(g) and sucrose crystallization temperatures (T(c)) of the composites. Sorption isotherms of the various sucrose/rbSt mixtures were determined gravimetrically with a controlled atmosphere microbalance (CAM) and verified by Karl Fischer analysis of selected samples. The CAM was also used to determine lag times and sucrose crystal growth rates by monitoring weight losses resulting from water liberation upon crystallization of sucrose at 23 degrees C. Results obtained by MDSC indicate that the T(c) increased linearly from approximately 110 degrees C for pure sucrose to approximately 140 degrees C with 20% rbSt at very low water content (<0.1%). Similarly, at 22% RH (4.4% H2O), T(c) increased from approximately 70 degrees C to 120 degrees C. In neither case was T(g) impacted significantly by increasing protein from 0 to 20%. No T(c) could be noted for samples with > or = 30% rbSt in nonisothermal conditions. Plasticization by water decreased both T(g) and T(c) quite similarly but didn't impact the noted effect of protein on T(c). Induction time for sucrose crystallization (i.e. nucleation) at approximately 45% RH (23 degrees C) increased almost 10-fold by addition of 10% rbSt, whereas rates of water loss due to crystallization decreased by no more than 2-3-fold. The overall results strongly indicate that formulations of higher protein concentration will be more resistant to sucrose crystallization and thus more robust when transiently exposed to storage temperatures above their T(g).

Absorption↗
PubMed1995

Impact of moisture on thermally induced denaturation and decomposition of lyophilized bovine somatotropin.

The nonisothermal transitions of lyophilized recombinant bovine somatotropin (rbSt) as seen via differential scanning calorimetry were evaluated with respect to moisture content. The transition peak temperature of rbSt decreased with increasing moisture from 161 degrees C in the dry state to a plateau of 65 degrees C at 28% moisture, which is similar to that of rbSt in solution. Using high performance liquid chromatography, this irreversible endothermic transition consisted primarily of unfolding, hydrophobic aggregation, and some covalent modifications. In the dry state, covalent modifications, including polymerization into compounds of higher molecular weight, were more prominent, while in the presence of moisture, hydrophobic aggregation was most prominent. The irreversibility and scan rate dependence of the endothermic phenomena supports the kinetic nature of the transition rather than a simple equilibrium between globular and unfolded states. The apparent activation energy for the net transition (i.e., unfolding, hydrophobic aggregation, and covalent modifications) was 57 kcal/mol for rbSt at 9.9% moisture. The observed enthalpy of the transition increased, decreased, then approximately leveled off as a function of increasing moisture content. This can be explained by the increasingly significant contribution of the exothermic aggregation at higher moisture contents.

Animals↗
PubMed1995

Thermally induced denaturation of lyophilized bovine somatotropin and lysozyme as impacted by moisture and excipients.

The endothermic thermal transitions (i.e., denaturation) of lyophilized recombinant bovine somatotropin (rbSt) and lysozyme as seen via differential scanning calorimetry were evaluated with respect to moisture and excipients. The denaturation temperature, Tm, of rbSt and lysozyme decreased with increasing moisture irrespective of the excipient. However, the magnitude of the decrease elicited by moisture was dependent on the type of excipient. Furthermore, the effect of the excipient was dependent on the moisture content; excipients decreased Tm in low moisture solids (i.e., < 5% moisture) and increased it in hydrated solids (i.e., > 15% moisture). In the dry state (< 1% moisture), the addition of 50% sucrose, sorbitol, or glycerol lowered the Tm of rbSt from 161 degrees C to 136, 120, and 83 degrees C, respectively, indicating a destabilizing mechanism. Likewise, the Tm of lysozyme decreased from 156 degrees C to 142, 128, and 97 degrees C due to the addition of sucrose, sorbitol, and glycerol, respectively. At higher moisture contents, the excipients promoted a higher transition temperature at a given moisture content than the pure protein systems, indicating a stabilizing mechanism. An increase in the enthalpy of unfolding for dehydrated lysozyme was noted with increasing levels of moisture and/or excipient, despite the observed decrease in Tm. The thermal stability, or Tm, of the dehydrated proteins appeared to be correlated to the glass transition temperature (Tg) of the excipient, which in turn should be related to the Tg of the system. The lower the Tg of the excipient, the greater was the degree of destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Estimation of recombinant bovine somatotropin solubility by excluded-volume interaction with polyethylene glycols.

PURPOSE: The potential to estimate protein solubilities, with limited protein, by excluded-volume interactions was evaluated using polyethylene glycols (PEG) and recombinant bovine Somatotropin (rbSt). METHODS: Solutions of rbSt were prepared at concentrations significantly below saturation solubility. Subsequently, varying amounts of PEG were added to force protein precipitation. Following centrifugation, the protein concentration in the supernatant was assayed by reversed-phase HPLC, where a logarithmic relationship between solubility and % PEG was observed. RESULTS: An apparent protein solubility in the absence of PEG was determined by extrapolation and compared well with values measured by conventional approaches. Slopes of log solubility versus % PEG curves were consistent with excluded-volume principles and depended on the molecular weight of the PEG used. Furthermore, the precipitation process proved to be reversible, allowing for recovery of intact protein. Solubility-pH profiles obtained in the presence of PEG greatly reduced the quantities of protein needed and compared favorably with profiles in the absence of PEG. CONCLUSIONS: Thus, it appears feasible and practical, with certain limitations, to obtain solubility estimates of proteins by volume-exclusion methods with limited supplies of protein.

Animals↗
PubMed1993

Cleansing enema prior to double-contrast barium enema examination: is it necessary?

In a prospective study, 443 patients referred for double-contrast barium enema examination were allocated to one of four regimens consisting of either 24 or 48 hours of clear liquids in combination with a cathartic laxative (magnesium sulfate), an irritant laxative (bisacodyl), and hydration. One regimen from each time group included a preliminary cleansing enema. Significantly higher bowel cleanliness scores were given to the 48-hour regimen with no cleansing enema (P < .0002). Scores for overall quality of the barium enema examination (based on detectability of a 1-cm lesion) showed no significant differences between a 24- and a 48-hour regimen, with or without a cleansing enema. No differences emerged in patient acceptance of the regimens, and 54%-57% of patients had no complaints about the preparation. The authors recommend a 48-hour preparation to minimize the risk of interfering fecal material, especially in subjects with colonic dysmotility. A time-consuming cleansing enema can be omitted.

Barium Sulfate↗
PubMed1978

Waardenburg's syndrome in Kenyan Africans.

Waardenburg's syndrome is characterized by deafness and pigment disorders of the eyes, hair and skin. Two types are distinguished; e.g. type I with dystopia canthorum and type II without dystopia canthorum. In Kenya 12 out of 724 pupils of schoools for the deaf were found to suffer from the syndrome. Ten of these belonged to eight families; 20 other members of these families had signs of the syndrome. Of these 30 patients 18 had type I, 12 type II. The expression of most characteristics in these Kenyan patients was almost the same as in Caucasians. The mode of inheritance was autosomal dominant, as in Caucasians. The interocular distances were measured in 168 healthy children and adults of the Luo tribe. In most age groups slightly larger values were found than in the Caucasian race. For the diagnosis of dystopia canthorum this should be taken into account.

Abnormalities, Multiple↗