Mannitol Crystallization Research Articles

Solute Crystallization in Mannitol–Glycine Systems—Implications on Protein Stabilization in Freeze‐Dried Formulations

The use of mannitol in combination with glycine has resulted in stable freeze‐dried protein formulations. Our objectives were to (1) study solute crystallization in ternary systems containing mannitol, glycine, and water during all the stages of freeze drying as a function of processing conditions and formulation variables; (2) investigate the effect of sodium phosphate buffer salts on the crystallization of both mannitol and glycine and vice versa; and (3) investigate the effects of these excipients in a freeze‐dried lactate dehydrogenase (LDH) formulation. X‐ray powder diffractometry (XRD) and differential scanning calorimetry (DSC) were used to study the frozen aqueous solutions. Phase transitions during primary and secondary drying were monitored by simulating the entire freeze‐drying process in situ in the sample chamber of the diffractometer. LDH activity after freeze drying was determined spectrophotometrically. In frozen aqueous solutions containing mannitol and glycine, each solute influenced the extent of crystallization of the other. The solutes crystallized as δ‐mannitol and β‐glycine during primary drying. Glycine had a stronger tendency to crystallize, while it was easier to influence mannitol crystallization. The buffer salts inhibited the crystallization of mannitol and glycine. However, in some cases, during primary drying, glycine crystallization was followed by that of disodium hydrogen phosphate dodecahydrate. The latter underwent dehydration forming an amorphous anhydrate. It was possible to correlate the extent of crystallization of mannitol and glycine in the lyophile with the retention of protein activity. An increase in buffer concentration decreased the crystallinity of mannitol and glycine. This translated to increased retention of protein activity. © 2003 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:2272–2283, 2003

Characterization of monoclinic crystals in tablets by pattern-fitting procedure using X-ray powder diffraction data

The purpose of this study is to characterize the monoclinic crystals in tablets by using X-ray powder diffraction data and to evaluate the deformation feature of crystals during compression. The monoclinic crystals of acetaminophen and benzoic acid were used as the samples. The observed X-ray diffraction intensities were fitted to the analytic expression, and the fitting parameters, such as the lattice parameters, the peak-width parameters, the preferred orientation parameter and peak asymmetric parameter were optimized by a non-linear least-squares procedure. The Gauss and March distribution functions were used to correct the preferred orientation of crystallites in the tablet. The March function performed better in correcting the modification of diffraction intensity by preferred orientation of crystallites, suggesting that the crystallites in the tablets had fiber texture with axial orientation. Although a broadening of diffraction peaks was observed in acetaminophen tablets with an increase of compression pressure, little broadening was observed in the benzoic tablets. These results suggest that “acetaminophen is a material consolidating by fragmentation of crystalline particles and benzoic acid is a material consolidating by plastic deformation then occurred rearrangement of molecules during compression”. A pattern-fitting procedure is the superior method for characterizing the crystalline drugs of monoclinic crystals in the tablets, as well as orthorhombic isoniazid and mannitol crystals reported in the previous paper.

Effective inhibition of mannitol crystallization in frozen solutions by sodium chloride.

The purpose of this work was to study the possibility of preventing mannitol crystallization in frozen solutions by using pharmaceutically acceptable additives. Differential scanning calorimetry (DSC) and low-temperature X-ray diffractometry (LTXRD) were used to characterize the effect of additives on mannitol crystallization. DSC screening revealed that salts (sodium chloride, sodium citrate, and sodium acetate) inhibited mannitol crystallization in frozen solutions more effectively than selected surfactants, alpha-cyclodextrin, polymers, and alditols. This finding prompted further studies of the crystallization in the mannitol-NaCl-water system. Isothermal DSC results indicated that mannitol crystallization in frozen solutions was significantly retarded in the presence of NaCl and that NaCl did not crystallize until mannitol crystallization completed. Low-temperature X-ray diffractometry data showed that when a 10% w/v mannitol solution without additive was cooled at 1 degrees C/min, the crystalline phases emerging after ice crystallization were those of a mannitol hydrate as well as the anhydrous polymorphs. In the presence of NaCl (5% w/v), mannitol crystallization was suppressed during both cooling and warming and occurred only after annealing and rewarming. In the latter case however, mannitol did not crystallize as the hydrate, but as the anhydrous delta polymorph. At a lower NaCl concentration of 1% w/v, the inhibitory effect of NaCl on mannitol crystallization was evident even during annealing at temperatures close to the Tg' (-40 degrees C). A preliminary lyophilization cycle with polyvinyl pyrrolidone and NaCl as additives rendered mannitol amorphous. The effectiveness of additives in inhibiting mannitol crystallization in frozen solutions follows the general order: salts > alditols > polyvinyl pyrrolidone > alpha-cyclodextrin > polysorbate 80 approximately polyethylene glycol approximately poloxamer. The judicious use of additives can retain mannitol amorphous during all the stages of the freeze-drying cycle.

Crystallization of mannitol below Tg' during freeze-drying in binary and ternary aqueous systems.

To characterize the phase transitions in a multicomponent system during the various stages of the freeze-drying process and to evaluate the crystallization behavior below Tg' (glass transition temperature of maximally freeze-concentrated amorphous phase) in frozen aqueous solutions and during freeze-drying. X-ray powder diffractometry (XRD) and differential scanning calorimetry (DSC) were used to study frozen aqueous solutions of mannitol with or without trehalose. By attaching a vacuum pump to the low-temperature stage of the diffractometer, it was possible to simulate the freeze-drying process in situ in the sample chamber of the XRD. This enabled real-time monitoring of the solid state of the solutes during the process. In rapidly cooled aqueous solutions containing only mannitol (10% w/w), the solute was retained amorphous. Annealing of frozen solutions or primary drying. both below Tg', resulted in crystallization of mannitol hydrate. Similar effects were observed in the presence of trehalose (2% w/w). At higher concentrations (> or =5% w/w) of this noncrystallizing sugar. annealing below Tg' led to nucleation but not crystallization. However, during primary drying, crystallization of mannitol hydrate was observed. The combination of in situ XRD and DSC has given a unique insight into phase transitions during freeze-drying as a function of processing conditions and formulation variables. In the presence of trehalose, mannitol crystallization was inhibited in frozen solutions but not during primary drying.

Crystallization behavior of mannitol in frozen aqueous solutions.

To study the effect of cooling rate, the influence of phosphate buffers and polyvinylpyrrolidone (PVP) on the crystallization behavior of mannitol in frozen aqueous solutions. Low-temperature differential scanning calorimetry and powder X-ray diffractometry were used to characterize the frozen solutions. Rapid cooling (20 degrees C/min) inhibited mannitol crystallization, whereas at slower cooling rates (10 degrees C and 5 degrees C/min) partial crystallization was observed. The amorphous freeze-concentrate was characterized by two glass transitions at -32 degrees C and -25 degrees C. When the frozen solutions were heated past the two glass transition temperatures, the solute crystallized as mannitol hydrate. An increase in the concentration of PVP increased the induction time for the crystallization of mannitol hydrate. At concentrations of > or =100 mM, the buffer salts significantly inhibited mannitol crystallization. The crystallization behavior of mannitol in frozen solutions was influenced by the cooling rate and the presence of phosphate buffers and PVP.

Strong and macroporous calcium phosphate cement: Effects of porosity and fiber reinforcement on mechanical properties.

Because of its excellent osteoconductivity and bone-replacement capability, self-setting calcium phosphate cement (CPC) has been used in a number of clinical procedures. For more rapid resorption and concomitant osseointegration, methods were desired to build macropores into CPC; however, this decreased its mechanical properties. The aims of this study, therefore, were to use fibers to strengthen macroporous CPC and to investigate the effects of the pore volume fraction on its mechanical properties. Water-soluble mannitol crystals were incorporated into CPC paste; the set CPC was then immersed in water to dissolve mannitol, producing macropores. Mannitol/(mannitol + CPC powder) mass fractions of 0, 10, 20, 30, and 40% were used. An aramid fiber volume fraction of 6% was incorporated into the CPC-mannitol specimens, which were set in 3 mm x 4 mm x 25 mm molds and then fractured in three-point flexure to measure the strength, work of fracture, and modulus. The dissolution of mannitol created well-formed macropores, with CPC at 40% mannitol having a total porosity of a 70.8% volume fraction. Increasing the mannitol content significantly decreased the properties of CPC without fibers (analysis of variance; p < 0.001). The strength (mean +/- standard deviation; n = 6) of CPC at 0% mannitol was 15.0 +/- 1.8 MPa; at 40% mannitol, it decreased to 1.4 +/- 0.4 MPa. Fiber reinforcement improved the properties, with the strength increasing threefold at 0% mannitol, sevenfold at 30% mannitol, and nearly fourfold at 40% mannitol. The work of fracture increased by 2 orders of magnitude, but the modulus was not changed as a result of fiber reinforcement. A scanning electron microscopy examination of specimens indicated crack deflection and bridging by fibers, matrix multiple cracking, and frictional pullout of fibers as the reinforcement mechanisms. Macroporous CPCs were substantially strengthened and toughened via fiber reinforcement. This may help extend the use of CPCs with macropores for bony ingrowth to the repair of larger defects in stress-bearing locations.

Formation of Macropores in Calcium Phosphate Cements through the Use of Mannitol Crystals

Formation of Macropores in Calcium Phosphate Cements through the Use of Mannitol Crystals

Protein formulation and lyophilization cycle design: prevention of damage due to freeze-concentration induced phase separation.

Hemoglobin has been previously shown to unfold during freeze drying when lyophilized from formulations that undergo freeze-concentration induced phase separation (Heller et al. 1997. Biotechnol Prog 13:590-596). In this report, we show that such damage may be avoided using kinetic strategies to arrest the phase separation. By rapidly cooling samples during liquid nitrogen spray-freeze drying, the time that the formulation spends in temperature regimes (ca. -3 to -23 degrees C) in which phase separation is both thermodynamically favorable and kinetically realizable is minimized. Increased protein damage with decreasing cooling rates and/or longer annealing periods at -7 degrees C is observed by FTIR spectroscopy. Phase separation and concomitant protein damage may also be avoided by addition of mannitol at concentrations sufficient to cause crystallization. Mannitol crystals segregate the freeze concentrated solution into microscopic domains that block propagation and nucleation of phase separating events. Addition of noncrystallizing sugars, such as sucrose and trehalose, or nonionic surfactants, such as Tween 80 and Triton X-100, has little protective effect against phase separation induced damage during freezing drying.

Characterization of Phase Transitions During Freeze-Drying by In Situ X-ray Powder Diffractometry

ABSTRACTThe purpose of this research was to develop the technique of in situ freeze-drying in the sample chamber of an X-ray powder diffractometer (XRD) and to monitor the phase transitions during the freeze-drying of aqueous solutions of sodium nafcillin (I) and mannitol (II). Aqueous solutions of I and II were frozen under controlled conditions in the sample chamber of an XRD. This variable temperature XRD was modified so that the sample chamber could be evacuated and the samples dried under reduced pressures. Thus the entire freeze-drying cycle was carried out in the XRD holder and the solid-state was monitored during the various stages of the process. Frozen solutions of I when annealed at −4°C resulted in crystallization of the solute as 'sodium nafcillin hydrate' (unknown stoichiometry). Primary drying at −10°C, resulted in partial dehydration to a poorly crystalline sodium nafcillin hemihydrate. There was no crystallization of mannitol when solutions of II were cooled and subjected to primary drying at −50°C. During the drying, the intensities of the characteristic X-ray lines of ice (d-spacings of 3.94, 3.70 and 3.48 A) were quantified. This enabled real time monitoring of the complete sublimation of crystalline ice. When the secondary drying was carried out at −25°C, mannitol crystallized as an anhydrous mixture of the &dL-and β-polymorphs. In a second set of experiments, the frozen solutions were warmed to −25°C and subjected to primary drying. Mannitol crystallized and its XRD pattern matched that of mannitol hydrate reported recently (Yu et al., Pharm. Res., 14s (1997) S-445). When the secondary drying was carried out at −10°C, there was no change in the XRD pattern suggesting the formation of a dehydrated hydrate. This in situ XRD technique enabled us to characterize the phase transitions during freeze-drying. It would be useful in developing a mechanistic understanding of the alterations in the solid-state during freeze-drying of complex, multi-component, pharmaceutical systems.

Formulations of Sugars with Amino Acids or Mannitol–Influence of Concentration Ratio on the Properties of the Freeze-Concentrate and the Lyophilizate

ABSTRACTFormulations consisting of either sucrose or trehalose with glycine, lysine–HCl, or mannitol were studied to determine how the ratio of the excipients affects the design of the lyophilization program and the properties of the final cake. Glass transitions (Tg, Tg), crystallization temperatures, and eutectic melting temperatures were measured by differential scanning calorimetry, the physical state of the excipients was determined by x-ray powder diffraction, and residual moisture was measured by Karl Fischer titration. The addition of increasing amounts of glycine, lysine–HCl, or mannitol to a sucrose solution caused a progressive depression of the Tg, an effect that was more pronounced with the amino acids. In equivalent ratios with sucrose, the two amino acids induced a comparableT'g shift due to their low Tg' values. For lysine–HCl, two apparent Tgwith midpoint temperatures of –69 and –56°C were measured. For mannitol, the T'gdepression was unexpected because mannitol exhibits a higher T'gthan that of sucrose. During lyophilization, the ratio of the amorphous amino acids or mannitol to the sugar determined whether crystallization could be induced by an annealing step performed after freezing. Crystallization could be verified by a shift of the formerly depressed T' back to the value of the sugar and by the detection of the eutectic melting peak of the crystallized compound. The crystallized excipients served as excellent bulking agents. In the freeze-dried cake, amorphous glycine and even more amorphous mannitol lowered the Tgvalue. If the cake was stored above Tg, subsequent crystallization of mannitol occurred. The results emphasize that the qualitative and quantitative composition of a formulation has profound implications on the design of a lyophilization program and on the characteristics of the freeze-dried cake.

Effect of Mannitol Crystallinity on the Stabilization of Enzymes during Freeze-Drying.

The stabilizing effect of mannitol during the freeze-drying of proteins was studied using L-lactate dehydrogenase (LDH, rabbit muscle), beta-galactosidase (Escherichia coli) and L-asparaginase (Erwinia chrysanthemi) as model proteins. Crystallization of mannitol was studied by powder X-ray diffraction and differential scanning calorimetry (DSC), in relation to the stabilizing effect. All the enzymes were protected concentration-dependently by amorphous mannitol, but the stabilizing effect was decreased with an increase in mannitol crystallinity. The heat-treatment of frozen solutions above crystallization temperature prior to drying enhanced mannitol crystallization and LDH inactivation. The importance of maintaining excipients in an amorphous state during freeze-drying, previously reported for Aspergillus oryzae beta-galactosidase (K. Izutsu et al., Pharm. Res., 10, 1233 (1993)), was confirmed using three different enzymes.

The effect of bulking agent on the solid-state stability of freeze-dried methylprednisolone sodium succinate.

The rate of hydrolysis of methylprednisolone sodium succinate in the freeze dried solid state at 40 degrees C was determined in the presence of two common bulking agents--mannitol and lactose--at two different ratios of drug to excipient. Residual moisture levels were less than 1% in all samples tested, with no significant difference in residual moisture among different formulations. Rate of hydrolysis was significantly higher in mannitol-containing formulations versus lactose-containing formulations, and the rate of hydrolysis increases with increasing ratio of mannitol to drug. Thermal analysis and x-ray diffraction data are consistent with a composition-dependent rate of crystallization of mannitol in the formulation and its subsequent effect on distribution of water in the freeze-dried matrix. Increased water in the microenvironment of the drug decreases the glass transition temperature of the amorphous phase, resulting in an increased rate of reaction. The physical state of lactose remained constant throughout the duration of the study, and the rate of hydrolysis was not significantly different from the control formulation containing no excipient. Thermal analysis and x-ray diffraction data are consistent with formation of a liquid crystal phase in freeze-concentrated solutions of methylprednisolone sodium succinate containing no excipient.

Identification of Some Sugars and Mannitol in Celery

SUMMARY— The carbohydrate content of celery petioles was determined using paper chromatographic techniques. Sucrose, glucose, fructose and mannitol were identified and quantitatively determined. Mannitol crystals were isolated. Sugars chromatographed with solvents containing boric acid showed characteristic stabilities to indicators.