聚乙二醇化蛋白质阳离子树脂色谱行为的研究

以溶菌酶(LYS)作为模式蛋白,对聚乙二醇化蛋白质在不同阳离子交换填料中色谱行为进行了比较研究。考察了不同分子质量的聚乙二醇化溶菌酶(PEG-LYS)在4种阳离子树脂中的动态载样量的差异,同时比较了不同上样量对纯度和洗脱盐浓度的影响。研究结果显示PEG修饰能降低蛋白质和离子交换树脂的结合力,且随着PEG的分子质量的增加,PEG-LYS动态载样量降低。对相同分子质量的PEG-LYS,产物纯度以及PEG-LYS的洗脱盐浓度随着上样量的增加而降低,研究结果显示,PEG分子质量和上样量对纯化工艺的研究有重要意义。     

Characterisation and physical stability of PEGylated glucagon

Glucagon was mono-PEGylated with PEG 5000 at Lys-12 to examine the effect on conformation and physical stability during purification and freeze-drying. The model peptide glucagon is highly unstable and readily forms fibrils in solution. Secondary structure was determined by FTIR and far-UV CD and physical stability was assessed by the Thioflavin T assay.

Glucagon samples were included, which underwent the same RP-HPLC purification and/or freeze-drying as glucagon–PEG 5000. After purification and freeze-drying glucagon samples showed formation of intermolecular β-sheet by FTIR, this correlated with shorter lag-times for fibrillation in the Thioflavin T assay. Formation of intermolecular β-sheet was less apparent for glucagon–PEG 5000 and no fibrillation was detected by Thioflavin T assay. Apparently PEGylation significantly improved the physical stability of glucagon after purification and freeze-drying, possibly by steric hindrance of peptide–peptide interactions.

Alterations in the secondary structure were observed for freeze-dried and reconstituted peptide samples by liquid FTIR. The peak for -helix shifted to 1664 cm⁻¹, which could possibly be explained by formation of 3₁₀-helix. Neither 3₁₀-helix nor intermolecular β-sheet could be detected by far-UV CD, where all peptide samples showed similar spectra.

In conclusion, glucagon–PEG 5000 showed a significantly improved physical stability during purification and freeze-drying compared to glucagon.     

Effect of PEG molecular weight and linking chemistry on the biological activity and thermal stability of PEGylated trypsin

PEGylated proteins are routinely used as therapeutics, but systematic studies of the effect of PEG molecular weight and linking chemistry on the biological activity and particularly the thermal stability of the conjugated protein are rarely made. Here, activated monomethoxypolyethylene glycol (mPEG)s (Mw 1100, 2000 and 5000 g/mol) were prepared using succinic anhydride (SA), cyanuric chloride (CC) or tosyl chloride (TC) and used to synthesise a library of trypsin conjugates. The enzyme activity (K_M, V_max and K_cat) of native trypsin and the mPEG-modified trypsin conjugates was compared using N-benzoyl-l-arginine p-nitroanilide (BAPNA) as a substrate, and their thermal stability determined using both BAPNA and N--benzoyl-l-arginine ethyl ester hydrochloride (BAEE) as substrates to measure amidase and esterase activity respectively. The effect of conjugate chemistry on trypsin autolysis was also examined at 40 °C. PEG-trypsin conjugates containing the higher molecular weight of mPEG (5000 g/mol) were more stable than free trypsin, and the conjugate containing CC-mPEG 5000 g/mol had the best thermal stability.     

Quantitation of free polyethylene glycol in PEGylated protein conjugate by size exclusion HPLC with refractive index (RI) detection

In this study, size exclusion high performance liquid chromatography was evaluated for its application in separation and quantitation of free polyethylene glycol (PEG) and its PEGylated-protein-conjugate (PEG-conjugate). Although the large mass of the free PEG (2-fold greater than the protein) made separation difficult, chromatographic conditions were identified enabling resolution and quantitation of the free PEG, PEG-conjugate and non-PEGylated protein with Shodex Protein KW803 and KW804 columns in series and refractive index detection. The optimum resolution of 1.7 and 2.0 was achieved for the free PEG and PEG-conjugate as well as the free PEG and non-PEGylated protein using 20 mM HEPES buffer at pH 6.5. Under this condition, the plot of log₁₀MW of all the pertinent analytes against retention time showed a linear relationship with a correlation coefficient of 1. Limited assay performance evaluation demonstrated that the method was linear in the concentration range of 10 to 250 μg/mL of free PEG with correlation coefficients of ≥0.99. When free PEG in this concentration range was spiked into PEG-conjugate samples at 1 mg/mL, the recovery was in the range of 78%–120%. Detection and quantitation limits were determined to be, respectively, 10 and 25 μg/mL for free PEG. The R.S.D. for intra- and inter-day precision was 0.09% or less for retention time measurements and 2.9% or less for area count measurements. Robustness testing was performed by deliberately deviating ±0.2 pH units away from the desired pH as well as by increasing the flow rate. These deviations resulted in no significant impact on area percent distribution of all species. However, separation was found to be sensitive to high ionic strength and buffer species.     

Microencapsulation of PEGylated Adenovirus within PLGA Microspheres for Enhanced Stability and Gene Transfection Efficiency

Purpose Green fluorescent protein (GFP) encoding adenovirus (ADV) was surface modified with polyethylene glycol (PEG) for microencapsulation within poly(lactic-co-glycolic acid) (PLGA) microspheres with the aim of improving stability and gene transfection activity. Methods A series of PEGylated ADV (PEG-ADV) with different PEG seeding densities on the viral surface was prepared and the GFP expression efficiency of each PEG-ADV in the series determined. The physical stabilities of naked ADV and PEG-ADV were comparatively evaluated by exerting a high shear homogenization process or by exposure to low pH. Naked ADV or PEG-ADV was microencapsulated within PLGA microspheres using a water-in-oil-in-water (W/O/W) double emulsion and solvent evaporation method. In vitro cumulative ADV and PEG-ADV release profiles from PLGA microspheres were determined over a 10-day period. GFP transfection efficiencies into HeLa cells were quantified, and the relative extent of the immune response for ADV and PEG-ADV encapsulated within PLGA microspheres was analyzed using macrophage cells. Results The physical stability of PEGylated ADV was greatly enhanced relative to that of naked ADV under the simulated W/O/W formulation conditions, such as exposure to an aqueous/organic interface during high shear-stressed homogenization. PEG-ADV was also more stable than ADV at low pH. ADV and PEG-AD were both released from PLGA microspheres similarly in a sustained fashion. However, when the ADV and PEG-ADV encapsulated microspheres transfected into HeLa cells, PEG-ADV microspheres demonstrated a higher GFP gene transfection efficiency than ADV microspheres. The PEG-ADV microspheres also exhibited a reduced extent of innate immune response for macrophage cells. Conclusions PEGylated ADV could be more safely microencapsulated within PLGA microspheres than naked ADV due to their enhanced physical stability under the harsh formulation conditions and acidic microenvironmental conditions of the microsphere, thereby increasing gene transfection efficiency.     

单链聚乙二醇化重组人干扰素ω的制备及其性质

采用直链PEG-琥珀酰亚胺琥珀酸酯 (mPEG-SS) 选择性修饰重组人干扰素ω (rhIFNω), 离子交换 色谱和凝胶过滤色谱组合分离纯化单链PEG修饰产物 (PEG-rhIFNω), 基质辅助激光解吸附飞行时间质谱(MALDI-TOF MS) 测定单链PEG-rhIFNω的相对分子量, 并利用RP-HPLC和SDS-PAGE对修饰产物进行分析。在优化的工艺条件下, 分离纯化收集液的单链PEG-rhIFNω, 平均含量达182 μg·mL⁻¹, 分离纯化收率超过22%, 纯度大于98%, SDS-PAGE法测得表观分子质量为60 810, MALDI-TOF MS法测得相对分子质量为43 790; 单链PEG-rhIFNω具有典型PEG修饰蛋白的特性, 抗病毒活性保留率为15.0%, 抗原性降低了64倍, 酸稳定性、抗 胰酶水解能力、血清稳定性和热稳定性均显著提高。单链PEG-rhIFNω的药学性质获得显著改善, 有望开发为 安全、长效的新型干扰素。     

聚乙二醇化尿酸酶体内外稳定性研究

目的研究聚乙二醇化尿酸酶体内外稳定性。方法以酶活为指标,考察聚乙二醇修饰尿酸酶和尿酸酶的温度稳定性(4~80℃)、酸碱稳定性、抗胰蛋白酶水解能力和小鼠体内半衰期。结果4~60℃条件下,修饰的尿酸酶的稳定性大于尿酸酶,在70℃时,两者活性均迅速降低。pH 5.2~6.0及pH 9.2~10.0之间,尿酸酶活性迅速降低,而修饰的尿酸酶却保留了较高的活性。抗胰蛋白酶水解中,尿酸酶在作用200 min后,活性降至最高值的20%;而修饰的尿酸酶仍保留70%的活性。体内稳定性试验表明,修饰的尿酸酶和尿酸酶的半衰期分别为1 530和45 min。结论聚乙二醇修饰可以增加尿酸酶的稳定性和抗胰蛋白酶水解能力,延长体内半衰期。     

The effect of deuterium oxide on the conformational stability and aggregation of bovine serum albumin

Abstract

Protein aggregation is a significant problem affecting the integrity of proteins, and is a major hindrance to the development of biopharmaceutical products. Deuterium oxide (D₂O), widely used in protein characterization studies, has been shown to promote protein aggregation when used as a substitute for water in most buffered protein solutions; however, a few studies have reported minor improvements in melting point temperatures for some proteins. Our study aims to investigate the effect of D₂O on protein stability, using bovine serum albumin (BSA) as a model. We performed accelerated stability studies at high temperatures and assessed the physical and conformational stability of BSA using fluorescence spectroscopy, dynamic light scattering (DLS) and size-exclusion high performance liquid chromatography. Our findings reveal that D₂O enhances the conformational stability of monomeric BSA, reducing monomer loss and formation of small aggregates at high temperatures. There is also an increase in the formation of larger aggregates probed by thioflavin T (ThT), however, the increase is not considered significant based on DLS results. Our findings demonstrate that exchanging water with D₂O can improve the stability of proteins in solution, by maintaining the stability of the monomeric form, which may be beneficial for the long-term storage of some biological products.     

N-terminal mono-PEGylation of growth hormone antagonist: Correlation of PEG size and pharmacodynamic behavior

Growth hormone antagonist (GHA), an analog of growth hormone (GH), can inhibit GH action and treat acromegaly. However, GHA suffers from a short plasma half-life of 15–20 min that has limited its clinical application. PEGylation, conjugation with polyethylene glycol (PEG), can increase the plasma half-life of GHA. Single PEG attachment (mono-PEGylation) at N-terminus of GHA has the advantages of product homogeneity and minimization of the bioactivity loss. Conjugation of large PEG molecule may increase the plasma half-life but could potentially decrease the bioactivity of GHA, due to the steric shielding effect of PEG. Thus, N-terminal mono-PEGylation of GHA with 20 kDa and 40 kDa PEG were used to look for a balance of the two competing factors. Sedimentation velocity analysis suggested that 40 kDa PEG was more efficient than 20 kDa PEG to elongate the molecular shape of the conjugate. As reflected by marginal suppression of insulin-like growth factor I (IGF-I), GHA conjugated with 40 kDa PEG was statistically indistinguishable from the saline solution that could not inhibit GH action. In contrast, GHA conjugated with 20 kDa PEG can apparently inhibit GH action, as reflected by IGF-I suppression of 30–43%. Thus, our work demonstrated the effective therapeutic potency of N-terminally mono-PEGylated GHA.     

Spray-dried indomethacin-loaded polyester nanocapsules and nanospheres: development, stability evaluation and nanostructure models

The industrial development of polymeric nanoparticle suspensions, as drug delivery systems, is limited due to the problems in maintaining stability of suspensions. In this work, a spray-drying technique was applied to dry nanocapsule and nanosphere suspensions prepared by nanoprecipitation of polyesters using SiO₂ as adjuvant. Powders obtained from nanocapsules presented stable drug recoveries and morphological characteristics after 5 months. For nanocapsules, nanostructures around 200 nm were observed by scanning electron microscopy (SEM) on the surface of microparticles of SiO₂, whereas for the nanosphere formulation, nanostructures with a reduced diameter (60–90 nm) were observed, despite the particle sizes of each original suspension being similar, when measured by photon correlation spectroscopy (PCS). In order to investigate the morphological aspects of nanocapsule and nanosphere powders, several nanosphere formulations were spray-dried using different concentrations of SiO₂ and a comparative study of the different colloidal systems (nanocapsules, nanospheres, nanoemulsion or nanodispersion) was carried out by PCS. SEM analyses showed that nanostructures with reduced diameter are formed independently of the adjuvant concentration. The dynamic properties of these systems allowed to suggest that the structure of the nanosphere particle (polymer, sorbitan monostearate and polysorbate 80) was a polymeric matrix dispersing the sorbitan monostearate which, when submitted to the spray-drying process in the presence of SiO₂, gave nanostructures presenting diameters around 80 nm covering the microparticles due to the release of lipophilic surfactant from the polymeric matrix.     

聚乙二醇修饰的钙黄绿素脂质体的制备与评价

目的:介绍冷冻融解法结合微滤膜挤出法制备聚乙二醇修饰的钙黄绿素脂质体的工艺。方法:用负染色法对其外观形态进行透射电镜观测,同时运用Zeta电势.粒径分析仪对超声波作用前后钙黄绿素脂质体的粒径分布及其Zeta电势进行检测。对超声波作用下脂质体内包覆的钙黄绿素的释放特性进行了评价。结果:制得的脂质体Zeta电势为负,其直径分布于80~285nm之间,平均直径为150nm。超声波作用后脂质体平均粒径减小,其内所包钙黄绿素快速释放。结论:表明超声波对脂质体内容物的释放具有明显的增强效果。     

Effect of Molecular Size of PEGylated Recombinant Human Epidermal Growth Factor on the Biological Activity and Stability in Rat Wound Tissue

The purpose of this study was to investigate the effect of size of polyethylene glycol (PEG) conjugated to recombinant human epidermal growth factor (rhEGF) on its stability in skin wound tissue and in vitro biological activity to find the desirable conjugate as topical therapeutic agent for wound healing. Site-specific PEGylation at N-terminus of rhEGF was performed with monomethoxy PEG-Butyraldehyde derivatives (MW 2, 5, and 20 kDa). Mono-PEG-rhEGFs retained 60–70% of biological activity of native rhEGF, and the effect of PEG size was not significant. The improvement of stability in the rat skin wound tissue was dependent on the increase of the PEG size attached. The degradation half-lives of native rhEGF, mono-PEG-2K-, ?5K-, and ?20K-rhEGFs were 1.1, 3.1, 5.2, and 41.5 hr, respectively. Therefore, mono-PEG-20K-rhEGF was considered to be the most desirable in terms of the increase of stability and the preservation of biological activity. This study suggests that the high molecular weight PEG at N-terminus of rhEGF would give a satisfactory stabilizing effect and thus may improve therapeutic efficacy in clinical use.     

Influence of PEGylation with linear and branched PEG chains on the adsorption of glucagon to hydrophobic surfaces

PEGylation has proven useful for prolonging the plasma half lives of proteins, and since approval of the first PEGylated protein drug product by the FDA in 1990, several PEGylated protein drug products have been marketed. However, the influence of PEGylation on the behavior of proteins at interfaces is only poorly understood. The aim of this work was to study the effect of PEGylation on the adsorption of glucagon from aqueous solution to a hydrophobic surface and to compare the effects of PEGylation with a linear and a branched PEG chain, respectively. The 3483 Da peptide glucagon was PEGylated with a 2.2 kDa linear and a branched PEG chain, respectively, and the adsorption behaviors of the three proteins were compared using isothermal titration calorimetry, fixed-angle optical reflectometry and total internal reflection fluorescence. PEGylation decreased the number of glucagon molecules adsorbing per unit surface area and increased the initial adsorption rate of glucagon. Furthermore, the results indicated that the orientation and/or structural changes of glucagon upon adsorption were affected by the PEGylation. Finally, from the isothermal titration calorimetry and the reflectometry data, it was observed that the architecture of the PEG chains had an influence on the observed heat flow upon adsorption as well as on the initial rate of adsorption, respectively.     

Adsorption of insulin with varying self-association profiles to a solid Teflon surface—Influence on protein structure, fibrillation tendency and thermal stability

Interfaces are present in the preparation of pharmaceutical products and are well known for having an influence on the physical stability of proteins. The aim of this study was to examine the conformation (i.e. secondary and tertiary structures) and fibrillation tendency, overall aggregation tendency and thermal stability of adsorbed human insulin at a solid particulate Teflon surface. The effects of changes in the association degree of insulin on the structure and stability have been determined. Using SEC-HPLC, association profiles were determined for insulin aspart, zinc-free human insulin and human insulin with two Zn²⁺ per hexamer in concentrations ranging from 0.1 mg/ml to 20 mg/ml. Insulin aspart was 100% monomeric, regardless of concentration. In contrast, human insulin went from 100% monomer to 80% hexamer, and 20% dimer/monomer and zinc-free human insulin from 100% monomer to 70% dimer and 30% monomer with increasing concentration. The secondary structure of the insulins changed upon adsorption, but only minor differences were observed among the insulins. Structural changes were observed when the insulin-surface ratio was varied, but at no point did the structure resemble that of fibrillated insulin in solution. The presence of particles resulted in increased fibrillation of human insulin. The lag-time of fibrillation decreased, when the amount of particles present was increased. In conclusion, the type and association degree of the three insulin variants has no major influence on the secondary structure observed after adsorption of insulin at the solid Teflon surface. However, the presence of particles increases the tendency of insulin to fibrillate.     

Evaluation of therapeutic potentials of site-specific PEGylated glucagon-like peptide-1 isomers as a type 2 anti-diabetic treatment: Insulinotropic activity, glucose-stabilizing capability, and proteolytic stability

PEGylation has been considered to be a good biotechnique for improving the therapeutic value of glucagon-like peptide-1 (GLP-1) analogs for the treatment of type 2 diabetes. Despite the attractive anti-diabetic potentials, GLP-1 does not exert its full biological action because of its extremely short life-time in vivo due to rapid proteolytic degradation. Here, the enzyme-resistant mono-PEGylated GLP-1 isomers substituted at Lys(26)- or Lys(34)-amine were prepared through a newly devised site-specific PEGylation process using a maleic anhydride-protection/deprotection method. The therapeutic potentials of these site-specific PEGylated GLP-1 isomers (Lys(26)- or Lys(34)-PEG-GLP-1) along with His(7)-(N-terminus) PEG-GLP-1 were evaluated by examining their insulinotropic activity, glucose-stabilizing capability, and proteolytic stability. Lys(34)-PEG-GLP-1 was found to have the well-preserved insulinotropic activity (93% efficacy versus GLP-1) in isolated rat pancreatic islets. Furthermore, Lys(34)-PEG-GLP-1 showed the most prominent glucose-stabilizing capability, evaluated via an oral glucose tolerance test in db/db mice by considering the following three crucial factors: (i) maximum blood glucose level (BGL), (ii) required time to lower the BGL below 100mg/dl, and (iii) total hypoglycemic degree. Additionally, Lys(34)-PEG-GLP-1 had longer half-lives than the other PEGylated GLP-1s in the dipeptidyl peptidase IV (DPP IV) inhibitor-treated liver or kidney homogenate, and its stability against DPP IV was also comparable to that of Lys(26)-PEG-GLP-1. Taken together, Lys(34)-PEG-GLP-1 displayed the promising characteristics in all evaluations versus His(7)- or Lys(26)-PEG-GLP-1. This site-specific PEGylated GLP-1 analog would have therapeutic usefulness for treating type 2 diabetes on account of the well-preserved insulinotropic activity, the increased proteolytic stability, and thereby the improved glucose-stabilizing capability.     

聚乙二醇化降纤酶对血小板聚集和凝血功能的影响

目的研究聚乙二醇化降纤酶对血小板聚集和凝血功能的影响。方法以二磷酸腺苷(ADP)诱导血小板聚集,用Labor aggregometer-153型双道血小板聚集仪测定血小板最大聚集率,观察聚乙二醇化降纤酶对大鼠体内血小板聚集的影响;同时通过对Beagle犬各个时间点凝血指标:凝血酶时间(TT)、凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)和纤维蛋白原(FIB)含量的观察,研究聚乙二醇化降纤酶对beagle犬凝血功能的影响。结果聚乙二醇化降纤酶低、中、高剂量组均能明显抑制ADP诱导的大鼠血小板聚集作用;降低纤维蛋白原(FIB)含量,延长beagle犬TT、PT、APTT时间,等剂量的聚乙二醇化降纤酶药效达峰和维持时间均较阳性药降纤酶的长。结论聚乙二醇化降纤酶具有较强的对抗血小板聚集和凝血作用。     

Effects of temperature and additives on stability and spectrum of a therapeutic fibroblast growth factor

Background and the purpose of the study

Human fibroblast growth factor 20 (FGF20) is a 16.5 kDa protein containing 154 amino acid residues with reportedly poor thermal stability, and low stability, which are considered to be major factors that can limit its pharmacological applications. Thus, the aim of this study was to enhance the thermal stability and bio activity of a therapeutic FGF20 by addition of sucrose or heparin as additives and also at different temperatures.

Methods

A variety of biophysical techniques such as far-UV circular dichroism (CD), fluorescence and high resolution derivative UV absorption spectroscopy, were employed to characterize FGF20 and study the effects of heparin and sucrose on its thermal stability and bio activity at pH 7.0.

Results

Results of this study suggest that human FGF20 is significantly unstable and induction of heat by increased temperatures results in aggregation and precipitation at pH 7.0. Great changes in the fluorescence intensity and shape were achieved by addition of heparin and sucrose at different temperatures compared to the control. From 10 °C to 60 °C, no significant changes were observed in far-UV CD spectrum compared to the control, but significant changes were observed by adding sucrose when these temperatures are above 45 °C. Upon addition of heparin and sucrose, the mitogenic activity increased significantly at all tested temperatures, and these changes may be related to the roles of heparin and sucrose on the structure and conformation of FGF20.

Conclusion

Results of this study suggest that heparin and sucrose as additives seems to benjm sufficient to prevent thermal inactivation of FGF20 and also maintain its conformation stability and bio activity.     

Aggregation and conformational stability evaluation of myoglobin in the presence of ionic surfactant

Sodium lauroyl sarcosinate (SLS) is frequently used for the solubilization of inclusion bodies in vitro due to its structural similarity to lipid plasma membrane. There are many factors that could influence protein aggregation propensity, including overall protein surface charge and hydrophobicity. Here, the aggregation pathway of myoglobin protein was studied under different conditions (pH 3.5 and 7.4) in the presence of varying concentrations of SLS to evaluate the underlying forces dictating protein aggregation. Data obtained from Rayleigh light scattering, ThT binding assay, and far-UV CD indicated that SLS have different effects on the protein depending on its concentration and environmental conditions. In the presence of low concentrations of SLS (0.05–0.1?mM), no aggregation was detected at both pH conditions tested. Whereas, as we reach higher SLS concentrations (0.5–10.0?mM), myoglobin started forming larger-sized aggregates at pH 3.5 and not pH 7.4. These results suggest that electrostatics interactions as well as hydrophobic forces play an important role in SLS-induced myoglobin aggregation.     

Investigation of PEG crystallization in frozen and freeze-dried PEGylated recombinant human growth hormone-sucrose systems: implications on storage stability

The objectives of the current study were to investigate (i) the phase behavior of a PEGylated recombinant human growth hormone (PEG-rhGH, ～60 kDa) during freeze-drying and (ii) its storage stability. The phase transitions during freeze-thawing of an aqueous solution containing PEG-rhGH and sucrose were characterized by differential scanning calorimetry. Finally, PEG-rhGH and sucrose formulations containing low, medium, and high polyethylene glycol (PEG) to sucrose ratios were freeze-dried in dual-chamber syringes and stored at 4°C and 25°C. Chemical decomposition (methionine oxidation and deamidation) and irreversible aggregation were characterized by size-exclusion and ion-exchange chromatography, and tryptic mapping. PEG crystallization was facilitated when it was covalently linked with rhGH. When the solutions were frozen, phase separation into PEG-rich and sucrose-rich phases facilitated PEG crystallization and the freeze-dried cake contained crystalline PEG. Annealing caused PEG crystallization and when coupled with higher drying temperatures, the primary drying time decreased by up to 51%. When the freeze-dried cakes were stored at 4°C, while there was no change in the purity of the PEG-rhGH monomer, deamidation was highest in the formulations with the lowest PEG to sucrose ratio. When stored at 25°C, this composition also showed the most pronounced decrease in monomer purity, the highest level of aggregation, and deamidation. Furthermore, an increase in PEG crystallinity during storage was accompanied by a decrease in PEG-rhGH stability. Interestingly, during storage, there was no change in PEG crystallinity in formulations with medium and high PEG to sucrose ratios. Although PEG crystallization during freeze-drying did not cause protein degradation, crystallization during storage might have influenced protein stability.     

光照对氧氟沙星葡萄糖注射液稳定性的影响

本文用十字交叉试验设计法考察ｐＨ值、光照强度和光照时间对氧氟沙星葡萄糖注射液稳定性的影响。结果表明：不同ｐＨ值的各组注射液，经强光照射后，其ｐＨ值、Ａ_４５０ｎｍ（有色物吸收值），对大肠杆菌［ＣＭＣＣ（Ｂ）４４１０３］的抗菌活性均没有明显改变（Ｐ＞０．０５），但氧氟沙星含量有所下降，杂质峰面积增加。后两者的变化受ｐＨ值影响最大，照射时间次之，光照强度影响最小。