Prakash Sundaramurthi

Glycine Crystallization in Frozen and Freeze-dried Systems: Effect of pH and Buffer Concentration

Purpose(1) To determine the effect of solution pH before lyophilization, over the range of 1.5 to 10, on the salt and polymorphic forms of glycine crystallizing in frozen solutions and in lyophiles. (2) To quantify glycine crystallization during freezing and annealing as a function of solution pH before lyophilization. (3) To study the effect of phosphate buffer concentration on the extent of glycine crystallization before and after annealing.Materials and MethodsGlycine solutions (10% w/v), with initial pH ranging from 1.5 to 10, were cooled to −50°C, and the crystallized glycine phases were identified using a laboratory X-ray source. Over the same pH range, glycine phases in lyophiles obtained from annealed solutions (0.25, 2 and 10% w/v glycine), were characterized by synchrotron X-ray diffractometry (SXRD). In the pH range of 3.0 to 5.9, the extent of glycine crystallization during annealing was monitored by SXRD. Additionally, the effect of phosphate buffer concentration (50 to 200xa0mM) on the extent of glycine crystallization during freezing, followed by annealing, was determined.ResultsIn frozen solutions, β-glycine was detected when the initial solution pH was ≥u20094. In the lyophiles, in addition to β- and γ-glycine, glycine HCl, diglycine HCl, and sodium glycinate were also identified. In the pH range of 3.0 to 5.9, decreasing the pH reduced the extent of glycine crystallization in the frozen solution. When the initial pH was fixed at 7.4, and the buffer concentration was increased from 50 to 200xa0mM, the extent of glycine crystallization in frozen solutions decreased with an increase in buffer concentration.ConclusionBoth solution pH and solute concentration before lyophilization influenced the salt and polymorphic forms of glycine crystallizing in frozen solutions and in lyophiles. The extent of glycine crystallization in frozen solutions was affected by the initial pH and buffer concentration of solutions. The high sensitivity of SXRD allowed simultaneous detection and quantification of multiple crystalline phases.

Crystallization of Trehalose in Frozen Solutions and its Phase Behavior during Drying

ABSTRACTPurpose(i) To study the crystallization of trehalose in frozen solutions and (ii) to understand the phase transitions during the entire freeze-drying cycle.MethodAqueous trehalose solution was cooled to −40°C in a custom-designed sample holder. The frozen solution was warmed to −18°C and annealed, and then dried in the sample chamber of the diffractometer. XRD patterns were continuously collected during cooling, annealing and drying.ResultsAfter cooling, hexagonal ice was the only crystalline phase observed. However, upon annealing, crystallization of trehalose dihydrate was evident. Seeding the frozen solution accelerated the solute crystallization. Thus, phase separation of the lyoprotectant was observed in frozen solutions. During drying, dehydration of trehalose dihydrate yielded a substantially amorphous anhydrous trehalose.ConclusionsCrystallization of trehalose, as trehalose dihydrate, was observed in frozen solutions. The dehydration of the crystalline trehalose dihydrate to substantially amorphous anhydrate occurred during drying. Therefore, analyzing the final lyophile will not reveal crystallization of the lyoprotectant during freeze-drying. The lyoprotectant crystallization can only become evident by continuous monitoring of the system during the entire freeze-drying cycle. In light of the phase separation of trehalose in frozen solutions, its ability to serve as a lyoprotectant warrants further investigation.

Calorimetric and Diffractometric Evidence for the Sequential Crystallization of Buffer Components and the Consequential pH Swing in Frozen Solutions

Sequential crystallization of succinate buffer components in the frozen solution has been studied by differential scanning calorimetry and X-ray diffractometry (both laboratory and synchrotron sources). The consequential pH shifts were monitored using a low-temperature electrode. When a solution buffered to pH < pK(a)(2) was cooled from room temperature (RT), the freeze-concentrate pH first increased and then decreased. This was attributed to the sequential crystallization of succinic acid, monosodium succinate, and finally disodium succinate. When buffered to pH > pK(a)(2), the freeze-concentrate pH first decreased and then increased due to the sequential crystallization of the basic (disodium succinate) followed by the acidic (monosodium succinate and succinic acid) buffer components. XRD provided direct evidence of the crystallization events in the frozen buffer solutions, including the formation of disodium succinate hexahydrate [Na(2)(CH(2)COO)(2).6H(2)O]. When the frozen solution was warmed in a differential scanning calorimeter, multiple endotherms attributable to the melting of buffer components and ice were observed. When the frozen solutions were dried under reduced pressure, ice sublimation was followed by dehydration of the crystalline hexahydrate to a poorly crystalline anhydrate. However, crystalline succinic acid and monosodium succinate were retained in the final lyophiles. The pH and the buffer salt concentration of the prelyo solution influenced the crystalline salt content in the final lyophile. The direction and magnitude of the pH shift in the frozen solution depended on both the initial pH and the buffer concentration. In light of the pH-sensitive nature of a significant fraction of pharmaceuticals (especially proteins), extreme care is needed in both the buffer selection and its concentration.

Influence of Crystallizing and Non-crystallizing Cosolutes on Trehalose Crystallization During Freeze-Drying

ABSTRACTPurposeTo study the influence of crystallizing and non-crystallizing cosolutes on the crystallization behavior of trehalose in frozen solutions and to monitor the phase behavior of trehalose dihydrate and mannitol hemihydrate during drying.MethodsTrehalose (a lyoprotectant) and mannitol (a bulking agent) are widely used as excipients in freeze-dried formulations. Using differential scanning calorimetry (DSC) and X-ray diffractometry (XRD), the crystallization behavior of trehalose in the presence of (i) a crystallizing (mannitol), (ii) a non-crystallizing (sucrose) solute and (iii) a combination of mannitol and a model protein (lactose dehydrogenase, catalase, or lysozyme) was evaluated. By performing the entire freeze-drying cycle in the sample chamber of the XRD, the phase behavior of trehalose and mannitol were simultaneously monitored.ResultsWhen an aqueous solution containing trehalose (4% w/v) and mannitol (2% w/v) was cooled to −40°C at 0.5°C/min, hexagonal ice was the only crystalline phase. However, upon warming the sample to the annealing temperature (−18°C), crystallization of mannitol hemihydrate was readily evident. After 3xa0h of annealing, the characteristic XRD peaks of trehalose dihydrate were also observed. The DSC heating curve of frozen and annealed solution showed two overlapping endotherms, attributed by XRD to the sequential melting of trehalose dihydrate—ice and mannitol hemihydrate—ice eutectics, followed by ice melting. While mannitol facilitated trehalose dihydrate crystallization, sucrose completely inhibited it. In the presence of protein (2xa0mg/ml), trehalose crystallization required a longer annealing time. When the freeze-drying was performed in the sample chamber of the diffractometer, drying induced the dehydration of trehalose dihydrate to amorphous anhydrate. However, the final lyophiles prepared in the laboratory lyophilizer contained trehalose dihydrate and mannitol hemihydrate.ConclusionsUsing XRD and DSC, the sequential crystallization of ice, mannitol hemihydrate, and trehalose dihydrate was observed in frozen solutions. Mannitol, by readily crystallizing as a hemihydrate, accelerated trehalose dihydrate crystallization in frozen solutions. However, by remaining amorphous, sucrose completely inhibited trehalose dihydrate crystallization. Crystallization of the lyoprotectantt in the model protein formulations might have serious implications on protein stability.

Phase Transitions in Frozen Systems and During Freeze–Drying: Quantification Using Synchrotron X-Ray Diffractometry

ABSTRACTPurpose(1) To develop a synchrotron X-ray diffraction (SXRD) method to monitor phase transitions during the entire freeze–drying cycle. Aqueous sodium phosphate buffered glycine solutions with initial glycine to buffer molar ratios of 1:3 (17:50xa0mM), 1:1 (50xa0mM) and 3:1 were utilized as model systems. (2) To investigate the effect of initial solute concentration on the crystallization of glycine and phosphate buffer salt during lyophilization.MethodsPhosphate buffered glycine solutions were placed in a custom-designed sample cell for freeze–drying. The sample cell, covered with a stainless steel dome with a beryllium window, was placed on a stage capable of controlled cooling and vacuum drying. The samples were cooled to −50°C and annealed at −20°C. They underwent primary drying at −25°C under vacuum until ice sublimation was complete and secondary drying from 0 to 25°C. At different stages of the freeze–drying cycle, the samples were periodically exposed to synchrotron X-ray radiation. An image plate detector was used to obtain time-resolved two-dimensional SXRD patterns. The ice, β-glycine and DHPD phases were identified based on their unique X-ray peaks.ResultsWhen the solutions were cooled and annealed, ice formation was followed by crystallization of disodium hydrogen phosphate dodecahydrate (DHPD). In the primary drying stage, a significant increase in DHPD crystallization followed by incomplete dehydration to amorphous disodium hydrogen phosphate was evident. Complete dehydration of DHPD occurred during secondary drying. Glycine crystallization was inhibited throughout freeze–drying when the initial buffer concentration (1:3 glycine to buffer) was higher than that of glycine.ConclusionA high-intensity X-ray diffraction method was developed to monitor the phase transitions during the entire freeze–drying cycle. The high sensitivity of SXRD allowed us to monitor all the crystalline phases simultaneously. While DHPD crystallizes in frozen solution, it dehydrates incompletely during primary drying and completely during secondary drying. The impact of initial solute concentration on the phase composition during the entire freeze–drying cycle was quantified.

Calorimetry and complementary techniques to characterize frozen and freeze-dried systems.

Lyophilization is a commonly used drying technique for thermolabile pharmaceuticals. Crystallization of formulation components may occur during various stages of the freeze-drying process. In frozen solutions, while crystallization of bulking agents is desirable, both from processing and product-elegance perspectives, buffer salt crystallization can cause a significant pH shift. Lyoprotectants (compounds that protect macromolecules, both during freeze-drying and subsequent storage) are effective only when retained amorphous. This review presents numerous applications of differential scanning calorimetry to characterize pharmaceutical systems in frozen state. These studies are aimed at defining the processing parameters and optimizing the freeze-drying cycle. Low temperature pH measurement and sub-ambient X-ray diffractometry served as excellent complementary tools in the characterization of frozen systems. The phase behavior of the systems during annealing (of frozen solutions), primary and secondary drying were monitored by X-ray diffractometry. Finally, the interplay of formulation composition and processing parameters on the development and optimization of freeze-drying cycles are reviewed.

The Effect of Crystallizing and Non-crystallizing Cosolutes on Succinate Buffer Crystallization and the Consequent pH Shift in Frozen Solutions

ABSTRACTPurposeTo effectively inhibit succinate buffer crystallization and the consequent pH changes in frozen solutions.MethodsUsing differential scanning calorimetry (DSC) and X-ray diffractometry (XRD), the crystallization behavior of succinate buffer in the presence of either (i) a crystallizing (glycine, mannitol, trehalose) or (ii) a non-crystallizing cosolute (sucrose) was evaluated. Aqueous succinate buffer solutions, 50 or 200xa0mM, at pH values 4.0 or 6.0 were cooled from room temperature to −25°C at 0.5°C/min. The pH of the solution was measured as a function of temperature using a probe designed to function at low temperatures. The final lyophiles prepared from these solutions were characterized using synchrotron radiation.ResultsWhen the succinic acid solution buffered to pH 4.0, in the absence of a cosolute, was cooled, there was a pronounced shift in the freeze-concentrate pH. Glycine and mannitol, which have a tendency to crystallize in frozen solutions, remained amorphous when the initial pH was 6.0. Under this condition, they also inhibited buffer crystallization and prevented pH change. At pH 4.0 (50xa0mM initial concentration), glycine and mannitol crystallized and did not prevent pH change in frozen solutions. While sucrose, a non-crystallizing cosolute, did not completely prevent buffer crystallization, the extent of crystallization was reduced. Sucrose decomposition, based on XRD peaks attributable to β-D-glucose, was observed in frozen buffer solutions with an initial pH of 4.0. Trehalose completely inhibited crystallization of the buffer components when the initial pH was 6.0 but not at pH 4.0. At the lower pH, the crystallization of both trehalose dihydrate and buffer components was evident.ConclusionWhen retained amorphous, sucrose and trehalose effectively inhibited succinate buffer component crystallization and the consequent pH shift. However, when trehalose crystallized or sucrose degraded to yield a crystalline decomposition product, crystallization of buffer was observed. Similarly, glycine and mannitol, two widely used bulking agents, inhibited buffer component crystallization only when retained amorphous. In addition to stabilizing the active pharmaceutical ingredient, lyoprotectants may prevent solution pH shift by inhibiting buffer crystallization.

Predicting the crystallization propensity of carboxylic acid buffers in frozen systems—relevance to freeze-drying

Selective crystallization of buffer components in frozen solutions is known to cause pronounced pH shifts. Our objective was to study the crystallization behavior and the consequent pH shift in frozen aqueous carboxylic acid buffers. Aqueous carboxylic acid buffers were cooled to -25°C and the pH of the solution was measured as a function of temperature. The thermal behavior of solutions during freezing and thawing was investigated by differential scanning calorimetry. The crystallized phases in frozen solution were identified by X-ray diffractometry. The malate buffer system was robust with no evidence of buffer component crystallization and hence negligible pH shift. In the citrate and tartarate systems, at initial pH <pKa2 , only the most acidic buffer component (neutral form) crystallized on cooling, causing an increase in the freeze-concentrate pH. Carboxylic acid buffers were rank ordered based on their propensity to crystallize in frozen solutions. From the aqueous solubility values of these carboxylic acids, which have been reported over a range of temperatures, it was also possible to estimate the degree of supersaturation at the subambient temperature of interest. This enabled us to predict their crystallization propensity in frozen systems. The experimental and the predicted rank orderings were in excellent agreement.

Thermophysical properties of carboxylic and amino acid buffers at subzero temperatures: relevance to frozen state stabilization.

Macromolecules and other thermolabile biologicals are often buffered and stored in frozen or dried (freeze-dried) state. Crystallization of buffer components in frozen aqueous solutions and the consequent pH shifts were studied in carboxylic (succinic, malic, citric, tartaric acid) and amino acid (glycine, histidine) buffers. Aqueous buffer solutions were cooled from room temperature (RT) to -25 °C and the pH of the solution was measured as a function of temperature. The thermal behavior of frozen solutions was investigated by differential scanning calorimetry (DSC), and the crystallized phases were identified by X-ray diffractometry (XRD). Based on the solubility of the neutral species of each buffer system over a range of temperatures, it was possible to estimate its degree of supersaturation at the subambient temperature of interest. This enabled us to predict its crystallization propensity in frozen systems. The experimental and the predicted rank orderings were in excellent agreement. The malate buffer system was robust with no evidence of buffer component crystallization and hence negligible pH shift. In the citrate and tartrate systems, at initial pH < pK(a)(2), only the most acidic buffer component (neutral form) crystallized on cooling, causing an increase in the freeze-concentrate pH. In glycine buffer solutions, when the initial pH was ∼3 units < isoelectric pH (pI = 5.9), β-glycine crystallization caused a small decrease in pH, while a similar effect but in the opposite direction was observed when the initial pH was ∼3 units > pI. In the histidine buffer system, depending on the initial pH, either histidine or histidine HCl crystallized.

Azithromycin Hydrates—Implications of Processing‐Induced Phase Transformations

According to label claims, in commercial solid dosage forms, azithromycin (AZI) exists either as a monohydrate (MH) or as a dihydrate (DH). Although these two forms are known to be relatively stable in the solid state, AZI sesquihydrate (SH) was observed in a drug product. This was believed to be a consequence of a processing-induced phase transformation. Our goal was to prepare and characterize AZI SH and map its solid-state transition pathways with other AZI phases. When dehydrated at temperatures <80°C, DH yielded an isomorphic dehydrate, whereas dehydration at ≥80°C yielded SH. Heating SH to 100°C or holding at 0% RH at room temperature, yielded an amorphous product through an intermediate isomorphic dehydrate, isostructural to SH. On the other hand, dehydration of MH (at ≥60°C) resulted in amorphization with no intermediate crystalline anhydrate. Diagnostic XRD peaks of AH, MH, SH, and DH enabled their unambiguous identification. However, the presence of crystalline excipients hindered active pharmaceutical ingredient characterization in drug product. Pattern subtraction method was used to selectively remove the contribution of the crystalline excipients to the overall diffraction pattern, thereby facilitating the physical characterization of AZI in the drug product.