Lysozyme stability in primary emulsion for PLGA microsphere preparation: effect of recovery methods and stabilizing excipients

Purpose. To investigate the conformational stability of a model protein, lysozyme, in the primary emulsion phase of the microsphere preparation process. Methods. The conformational stability of lysozyme during primary emulsification was assessed by differential scanning calorimetry (DSC) and enzymatic activity assay. PEG 400 was used to separate lysozyme from water-in-oil (w/o) emulsion containing poly(lactide-co-glycolide) (PLGA). Results. No significant changes in the recovery of lysozyme were observed due to increasing sonication time from 20 to 60 s at 40 W or increasing intensity from 40 to 60 W for 20 s. By using the method involving PEG 400, lysozyme recovery in the presence of PLGA was increased from 11.8% to 70%. Hydroxypropyl--cyclodextrin (HP--CD) increased lysozyme recovery from 35% to 70% at low lysozyme concentration (20 mg/ml), and from 70% to 77% at high lysozyme concentration (100 mg/ml) in the presence of PLGA. Sugars such as trehalose and mannitol failed to increase lysozyme recovery. DSC results suggested the retention of the conformational structure of the recovered lysozyme, which was supported by an enzymatic activity assay. Conclusions. HP--CD was found to be a promising stabilizer that protected lysozyme during the primary emulsification. Protein recovery method with the help of PEG 400 allowed the study of protein stability in w/o emulsions in the presence of PLGA. DSC provided supplementary information on the conformational changes of lysozyme during emulsification.     

A scanning calorimetric study of the effect of clover saponin on liposomal phospholipid membrane

The effect of clover saponin on the phase transition of liposomal lipid bilayers of dimyristoyl phosphatidylcholine was investigated with differential scanning calorimetry. The thermograms of the liposomal bilayers incorporated with the clover saponin were obtained, and the enthalpy changes and the sizes of cooperative unit of the transition were calculated. The results showed that incorporation of the clover saponin into the liposomal bilayers effectively reduced the transition temperature at which the transition from solid state to liquid-crystalline state occurs, and broadened the thermogram peaks. It also reduced the size of cooperative unit of the transition. These results indicate that the clover saponin might have significant effect on the fluidity of biological membranes.     

薄层双波长扫描法测定东莨菪碱贴膜的化学稳定性

本文建立了东莨菪碱贴膜化学稳定性的薄层双波长扫描定量测定方法。以氯仿—甲醇—氨水(8.5:1.5:0.07)溶剂系统展开硅胶G薄层板上样品,经碘化铋钾试液显色,采用反射法锯齿扫描,测定了初均速条件下东莨菪碱贴膜的贮存期T_0.9~(25)=1.65a,降解活化能Ea=21.213kcal/mol。     

盐酸山莨菪碱对头孢拉定注射液稳定性的影响

目的:研究盐酸山莨菪碱(654-2)对头孢拉定(Cep)注射液稳定性的影响.方法:采用反相高效液相色谱法测定3种温度F,不同时间的Cep在生理盐水和含654-2盐水中的含量.结果:两组数据经统计学处理无显著性差异.结论:Cep与654-2配伍时其含量稳定.     

美托拉宗片剂中药物多晶现象的研究

目的测定美托拉宗原研片(规格:0.5mg)的晶型。方法通过差示扫描量热法(DSC)和粉末X射线衍射法(PXRD)分别对美托拉宗原料药、原研片、无定形样品、载药分别为1,5和50mg自制片进行检测与分析。结果经检测原料药药物晶型为晶型Ⅰ,原研片存在微弱的晶型Ⅰ熔融峰,无定形在检测过程中吸热发生晶型转变,转变成稳定的晶型Ⅰ,无定形自制片剂未发现晶型I熔融峰,而采用原料药所制备的载药片剂均检出明显的晶型Ⅰ熔融峰。结论原研片中药物的晶型为晶型Ⅰ。     

The Cryopreservation of Liposomes. 1. A Differential Scanning Calorimetry Study of the Thermal Behavior of a Liposome Dispersion Containing Mannitol During Freezing/Thawing

The thermal behavior of water in liposome dispersions and in liposome dispersions containing mannitol at subzero temperatures was investigated with differential scanning calorimetry (DSC). The cooling curves from 20 down to - 60°C for a liposome dispersion (bilayer composition PL100H/DCP), monitored at cooling rates of 5 and 10°C/min, showed several heat flows related to water crystallization. All lipid-containing dispersions showed water crystallization at temperatures below –40°C. The magnitude of this heat flow strongly depended on the experimental variables. Cooling rate, particle size, lipid concentration, and location and nature of the cryoprotectant all influenced the water crystallization behavior as shown in the DSC cooling curve. Different fractions of water–presumably related to their location in the dispersion–could be distinguished. It is concluded that DSC provides a valuable tool for the detection of changes in the physical state of water in liposome dispersions during freezing/thawing. The insights gained from these DSC studies may make it possible to select–on the basis of rational considerations rather than by trial and error–optimum conditions for the cryopreservation of liposomes containing water-soluble drugs.     

Chemical and Thermal Stability of Insulin: Effects of Zinc and Ligand Binding to the Insulin Zinc-Hexamer

Purpose To study the correlation between the thermal and chemical stability of insulin formulations with various insulin hexamer ligands.Materials and Methods The thermal stability was investigated using differential scanning calorimetry (DSC) and near-UV circular dichroism (NUV-CD). The formation of chemical degradation products was studied with reversed-phase and size-exclusion chromatography and mass spectrometry.Results An excellent correlation between the thermal stabilization by ligand binding and the deamidation of Asn^B3 was observed. The correlation between thermal stability and the formation of covalent dimer and other insulin related products was less clear. Zinc was found to specifically increase the deamidation and covalent dimer formation rate when the insulin hexamer was not further stabilized by phenolic ligand. Thiocyanate alone had no effect on the thermal stability of the insulin zinc-hexamer but significantly improved the chemical stability at 37°C. At low temperatures thiocyanate induced a conformational change in the insulin hexamer. NUV-CD thermal scans revealed that this effect decreased with temperature; when the thermal denaturation temperature was reached, the effect was eliminated.Conclusions Thermal stability can be used to predict the rate of Asn^B3 deamidation in human insulin. Chemical degradation processes that do not rely on the structural stability of the protein do not necessarily correlate to the thermal stability.     

采用差示扫描量热技术建立苦参碱纯度测定新分析方法

目的研究采用差示扫描量热技术（DSC）,建立中药活性成分苦参碱化学样品纯度的新检测分析方法。方法针对苦参碱化学样品,考察了溶剂残留、炉体气氛、升温速率、称样量四个因素对差示扫描量热法检测结果的影响;优化并确定了最佳检测条件,测定了苦参碱化学样品的纯度值;利用高效液相色谱法（HPLC）对建立的方法及检测结果进行了验证。结果本研究建立的差示扫描量热法进行苦参碱样品纯度检测具有较高的科学性和可行性,最佳实验条件为升温速率4.0K/min,称样量为2.5mg～3.7mg,炉内气体为静态空气,经检测获得苦参碱样品的化学纯度值为99.818%;高效液相色谱法测得苦参碱样品化学纯度值为99.825%。结论利用建立的差热扫描量热分析方法可以快速、准确地测定苦参碱化学纯度,论文研究为苦参碱纯度测定提供了一种新分析技术和方法。     

Application of hanging drop technique to optimize human IgG formulations

Objectives The purpose of this work is to assess the hanging drop technique in screening excipients to develop optimal formulations for human immunoglobulin G (IgG). Methods A microdrop of human IgG and test solution hanging from a cover slide and undergoing vapour diffusion was monitored by a stereomicroscope. Aqueous solutions of IgG in the presence of different pH, salt concentrations and excipients were prepared and characterized. Key findings Low concentration of either sodium/potassium phosphate or McIlvaine buffer favoured the solubility of IgG. Addition of sucrose favoured the stability of this antibody while addition of NaCl caused more aggregation. Antimicrobial preservatives were also screened and a complex effect at different buffer conditions was observed. Dynamic light scattering, differential scanning calorimetry and size exclusion chromatography studies were performed to further validate the results. Conclusions In conclusion, hanging drop is a very easy and effective approach to screen protein formulations in the early stage of formulation development.     

Use of Microcalorimetry and Its Correlation with Size Exclusion Chromatography for Rapid Screening of the Physical Stability of Large Pharmaceutical Proteins in Solution

The utility of microcalorimetry as a rapid screening tool for assessing the solution stability of high molecular weight pharmaceutical proteins was evaluated by using model recombinant antibodies, Protein I and Protein II. Changes in the transition midpoint, T_m, were monitored as a function of pH and/or in the presence of excipients, and results were compared with traditional accelerated stability data from samples that were analyzed by size exclusion chromatography (SEC). The data from microcalorimetry were well correlated with those from SEC for predicting both optimal solution pH as well as excipient effects on solution stability. These results indicate that microcalorimetry can be an efficient screening tool useful in identifying optimal pH conditions and excipients to stabilize pharmaceutical proteins in solution formulations.     

差示扫描量热法测定磺胺类化学对照品的纯度

目的 采用差示扫描量热法(differential scanning calorimetry,DSC)测定磺胺类化学对照品纯度。方法 采用优化DSC实验参数,载气流速50 mL·min^-1,升温速率3 ℃·min^-1,称样量1.3~2.0 mg,对7种磺胺类化学对照品进行纯度分析。结果 DSC测得磺胺噻唑纯度为99.9%,磺胺甲二唑为99.9%,磺胺甲氧嗪为99.8%,磺胺嘧啶为99.8%,磺胺甲噁唑为99.9%,磺胺多辛为99.5%和磺胺二甲嘧啶为99.9%,其结果与质量平衡法测定结果基本一致。结论 DSC简便、快捷、无需标准物质,为磺胺类化学对照品纯度测定提供了新的检测方法。     

The Effect of Additives on the Crystallization of Cefazolin Sodium During Freeze-Drying

Purpose. To monitor the phase transitions during freeze-drying of cefazolin sodium (I) as a function of process and formulation variables. Methods. Aqueous solutions of I were frozen under controlled conditions in the sample chamber of a variable temperature X-ray powder diffractometer (XRD). The instrument was modified so that the chamber could be evacuated and the samples dried under reduced pressures. Thus, the entire freeze-drying process was carried out in the XRD holder with real time monitoring of the phase transitions during the different stages of freeze-drying. Results. When aqueous solutions of cefazolin sodium (10% w/w) were cooled to -40°C, the XRD pattern revealed only the crystallization of ice. Annealing the frozen sample led to the crystallization of I as the pentahydrate. Differential scanning calorimetry revealed that the presence of isopropyl alcohol (IPA) (5% w/w) led to a decrease in the Tg, the glass transition temperature of the system, and lowered the temperature of crystallization. The crystallization was studied at -8 and at -15°C in the XRD, and, as expected, more rapid crystallization was observed at the higher temperature. Primary drying at -8°C led to the dehydration of the pentahydrate, resulting in a poorly crystalline product. Again, XRD permitted real time monitoring of the decrease in intensities of some characteristic peaks of the pentahydrate. The in situ XRD technique also enabled us to study the effects of processing conditions (different primary and secondary drying temperatures) and crystalline bulking agents on the solid-state of I in the lyophile. When I was lyophilized using mannitol or glycine as an additive, without an annealing step, the drug was X-ray amorphous although the additive crystallized. When annealed and freeze-dried, I remained crystalline in the presence of glycine but not in the presence of mannitol. Conclusions. The in situ XRD technique has enabled us to characterize the phase transitions during freeze-drying of cefazolin sodium in multicomponent systems.     

基于三种原理分离技术的多肽类药物比伐芦定杂质分析

摘要：目的 建立不同分离原理的高效液相色谱法,对7家企业的比伐芦定原料药中相关杂质进行分析,为全面控制比伐芦定有关物质提供依据。方法 采用反相高效液相色谱法（RP-HPLC）对11个杂质进行分离分析;采用亲水色谱法（HILIC-HPLC）对4个工艺杂质进行控制;采用分子排阻色谱法（SEC-HPLC）对聚合物进行测定。结果 建立的RP-HPLC方法可将主成分及11个杂质有效分离,11个杂质的校正因子均在0.8～1.2之间,比伐芦定检出浓度为0.1μg·ml-1,检测限为0.004%;建立的HILIC-HPLC方法可对主成分及4个工艺杂质有效分离,比伐芦定检出浓度为0.3μg·mL-1,检测限为0.01%;在SEC-HPLC条件下聚合物和比伐芦定依次出峰,两者分离度为2.9,比伐芦定检出浓度为6ng·mL-1,检测限为0.0006%。采用主成分自身对照法计算7家企业15批样品中15种已知杂质及聚合物,不同企业的产品杂质含量与其生产工艺存在一定的相关性。结论 三种不同原理的方法均专属性良好,灵敏度高,耐用性好,结果可靠,可用于比伐芦定有关物质的质量控制。     

A Calorimetric Method to Estimate Molecular Mobility of Amorphous Solids at Relatively Low Temperatures

Purpose To present a calorimetry-based approach for estimating the initial (at the onset of annealing) relaxation time (τ ⁰) of organic amorphous solids at relatively low temperatures, and to assess the temperature where molecular mobility of the amorphous drug is reduced to a level comparable with the desired shelf-life of the product.Materials and Methods Values of τ ⁰ for six amorphous pharmaceutical compounds were estimated based on the nonlinear Adam–Gibbs equation. Fragility was determined from the scanning rate-dependence of the glass transition temperature (T _g). The initial enthalpic and entropic fictive temperatures were obtained from the T _g and the heat capacities (C _p) of the amorphous and crystalline forms.Results At a relatively low temperature (∼40°C or more below T _g), τ ⁰ for the different compounds varies by over an order of magnitude. For some materials, the practical storage temperature at T _g − 50 K was found to be still too high to ensure long-term stability. The estimated τ ⁰ is highly sensitive to the fragility of the material and the C _p of the crystalline and amorphous forms. Materials with high fragility or greater C _p differences between crystalline and amorphous forms tend to have longer τ ⁰.Conclusions The proposed method can be used to estimate molecular mobility at relatively low temperatures without having to conduct enthalpy recovery experiments. An accurate τ ⁰ determination from this method relies on faithful fragility measurements.     

Lipophilic derivatization of synthetic peptides belonging to NS3 and E2 proteins of GB virus‐C (hepatitis G virus) and its effect on the interaction with model lipid membranes

Abstract: The synthesis by solid‐phase methodologies of peptides belonging to structural and non‐structural proteins of GB virus C as well as its N‐α‐acylation with myristate and palmitate fatty acids is described. To explore the peptide–lipid interactions we have used liposomes composed of dipalmitoylphosphatidylcholine as model membranes and complementary spectroscopic and calorimetric techniques. Our results show that structural and more clearly the structural lipophilic peptide sequences incorporated into lipid bilayers perturb the packing of lipids and affect their thermotropic properties, more than the non‐structural selected sequence. However, the binding of the synthetic sequences to lipid membranes occurred without any restructuration of the peptides.     

Physical characterisation of formulations for the development of two stable freeze-dried proteins during both dried and liquid storage

The development of stable freeze-dried proteins requires maintaining the physical and biological integrity of the protein as well as increasing the efficiency of the manufacturing process. Our objective was to study the effects of various excipients on both the physical characterisation and the dried and liquid stability of two proteins. Thermo-physical properties of 13 formulations were determined using both differential scanning calorimetry and freeze–drying microscopy. The antigenic activity was evaluated immediately after freeze–drying and after subsequent storage in both dried and liquid state. From the comparison between glass transition (T′_g) and collapse (T_coll) temperatures, we concluded that the collapse temperature was a more relevant parameter than T′_g for freeze–drying cycle development and optimisation. One crystalline formulation composed of 4% mannitol and 1% of sucrose protected efficiently both proteins during subsequent storage in dried state (6 months at 25 °C) and in liquid state (3 months at 4 °C after rehydration). However, the freeze–drying behaviour of this crystalline formulation remained difficult to predict and control. On the other hand, two amorphous formulations composed of 4% of maltodextrin and 0.02% of Tween 80, or 5% of BSA preserved antigenic activity during storage in dried state. The glassy character of these formulations as well as their high collapse temperature values (−9 and −12 °C, respectively) should allow simplification and shortening of freeze–drying process.     

Comparison of the interaction of methionine and norleucine-containing peptides with phospholipid bilayers

Norleucine is a structural analog of methionine with a methylene group replacing the thio ether. Despite the close structural similarity of these two amino acids, norleucine-containing peptides have markedly different behaviour with phospholipids compared with methionine-containing peptides. For example, HCO-L-Ahx-L-Leu-L-Phe-OMe behaves as a hydrophobic peptide when mixed with dimyristoylphosphatidylcholine. This peptide lowers the enthalpy of the lipid phase transition. The effect is independent of the rate of heating. With the homologous peptide, HCO-L-Met-L-Leu-L-Phe-OMe, the results are markedly dependent on scan rate with a higher enthalpy observed at faster scan rates. Only at a scan rate of 0.2 K min^-1 do the two peptides approach similar behaviour. The higher enthalpy observed for samples with the methionine peptide at higher scan rates can be explained assuming that the peptide aggregates at low temperature. As the phase transition temperature is approached, the more hydrophilic methionine peptide partitions more slowly into the membrane than the norleucine peptide. Partitioning of the peptides between aqueous and lipid phases was measured at 37° by centrifuging down the lipid-bound fraction. At a peptide concentration of 15 μM and a lipid concentration of 1.4 mM, 89% of the HCO-L-Ahx-L-Leu-L-PheOMe and 97% of the HCO-L-Met-L-Leu-L-PheOMe remained in the supernate, indicating a greater tendency of the norleucine-containing peptide to partition into the lipid phase. The peptides Ac-L-Phe-L-Met-L-Arg-L-Phe-NH₂ and Ac-L-Phe-L-Ahx-L-Arg-L-Phe-NH₂ are readily soluble in water. These peptides were deposited with the lipid from a solution in chloroform/methanol. After repeated scanning and/or incubation at the phase transition temperature the effect of the peptide in raising the phase transition temperature of the lipid was lost. Thus these peptides were no longer incorporated into the bilayer after these treatments. The loss of the alterations of the phase transitions caused by these peptides was observed upon immediate rescanning of the methionine-containing samples while for the norleucine-containing ones, incubation for several hours at the phase transition was required. Our results demonstrate that norleucine-containing peptides partition more rapidly into the lipid phase while the methionine peptides are more rapidly extracted from the lipid into the aqueous phase. The results emphasize the importance of determining the effect of scan rate as a criterion of the attainment of equilibrium in lipid-peptide mixtures. We demonstrate how relatively minor changes in structure can alter the behaviour of peptides with lipids. Our results suggest that the pharmacodynamics of peptides in which norleucine has replaced methionine will be altered.     

Characterisation of the Glass Transition of an Amorphous Drug Using Modulated DSC

Purpose. The use of modulated differential scanning calorimetry (MDSC) as a novel means of characterising the glass transition of amorphous drugs has been investigated, using the protease inhibitor saquinavir as a model compound. In particular, the effects of measuring variables (temperature cycling, scanning period, heating mode) have been examined. Methods. Saquinavir samples of known moisture content were examined using a TA Instruments 2920 MDSC at a heating rate of 2°C/min and an amplitude of ± 0.159°C with a period of 30 seconds. These conditions were used to examine the effects of cycling between - 50°C and 150°C. A range of periods between 20 and 50 seconds were then studied. Isothermal measurements were carried out between 85°C and 120°C using an amplitude of ± 0.159°C with a period of 30 seconds. Results. MDSC showed the glass transition of saquinavir (0.98 ± 0.05%w/w moisture content) in isolation from the relaxation endotherm to give an apparent glass transition temperature of 107.0° C ± 0.4C. Subsequent temperature cycling gave reproducible glass transition temperatures of approximately 105°C for both cooling and heating cycles. The enthalpic relaxation peak observed in the initial heating cycle had an additional contribution from a Tg 'shift' effect brought about by the difference in response to the glass transition of the total and reversing heat flow signals. Isothermal studies yield a glass transition at 105.9°C ± 0.1°C. Conclusions. MDSC has been shown to be capable of separating the glass transition of saquinavir from the relaxation endotherm, thereby facilitating measurement of this parameter without the need for temperature cycling. However, the Tg 'shift' effect and the number of modulations through the transition should be taken into account to avoid drawing erroneous conclusions from the experimental data. MDSC has been shown to be an effective method of characterising the glass transition of an amorphous drug, allowing the separate characterisation of the Tg and endothermic relaxation in the first heating cycle.     

A Central Composite Design to Investigate the Thermal Stabilization of Lysozyme

Purpose. The formulation and processing of protein drugs requires the stabilization of the native, biologically active structure. Our aim was to investigate the thermal stability of a model protein, lysozyme, in the presence of two model excipients, sucrose and hydroxypropyl--cyclodextrin (HP--CD). Methods. We used high sensitivity differential scanning calorimetry (HSDSC) in combination with a central composite design (CCD). As indicators of protein thermal stability, the measured responses were the unfolding transition temperature (T_m), the onset temperature of the denaturation (T₀), and the extrapolated onset temperature (T_o,e). Results. A highly significant (F probability <0.001) statistical model resulted from analysis of the data. The largest effect was due to pH (over the range 3.2-7.2), and the pH value that maximized T_m was 4.8. Several minor but significant effects were detected that were useful for mechanistic understanding. In particular, the effects of protein concentration and cyclodextrin concentration on T_m and T_o,e were found to be pH-dependent. This was indicative of the partially hydrophilic nature of protein-protein interactions and protein-cyclodextrin interactions, respectively. Conclusions. Response surface methodology (RSM) proved efficient for the modeling and optimization of lysozyme thermal stability as well as for the physical understanding of the protein-sugar-cyclodextrin system in aqueous solution.