Effects of Excipient Interactions on the State of the Freeze-Concentrate and Protein Stability

Purpose

The physical state of excipients in freeze-dried formulations directly affects the stability of the active pharmaceutical ingredient (API). Crystallization of trehalose and mannitol in frozen solutions has been shown to be a function of composition. However, to date a detailed study of the effect of concentrations of the API and other excipients on the crystallinity of mannitol and trehalose in frozen solutions has not been reported.

Methods

The crystallinity of mannitol and trehalose in frozen solutions was characterized by Differential Scanning Calorimetry, X-ray diffractometry, and FTIR spectroscopy. The secondary structure of BSA was probed by FTIR, and Circular Dichroism spectroscopy in frozen and thawed solutions, respectively.

Results

Trehalose crystallization was accompanied by unfolding of BSA. BSA delayed and reduced the extent of mannitol and trehalose crystallization. Similar effects were observed upon adding D₂O (≥5% w/w) and low concentrations of polysorbate 20 (≤0.2% w/w) with retention of BSA in its native conformation. At high BSA to trehalose mass ratio, the protein could stabilize itself in the frozen state, but unfolded upon thawing.

Conclusions

The API and other excipients, in a concentration-dependent manner, influenced the physical state of the freeze concentrate as well as the stability of the API.

     

微囊化工艺和相关辅料对蛋白类药物稳定性的影响

蛋白类药物微球的制备工艺可显著影响其结构功能(生物活性)的稳定性.本研究考察了微囊化过程中不同工艺、辅料、有机溶媒等因素对重组牛生长激素稳定性的影响,为优化重组牛生长激素微球注射剂工艺提供依据.     

辅料对碘昔兰注射液高温灭菌稳定性的影响

目的考察不同辅料对国产碘昔兰注射液高温灭菌稳定性的影响。方法测定120℃高温灭菌前后8组含不同辅料的碘昔兰注射液的pH值、游离碘离子浓度、色谱纯和含量变化。结果同时含三羟甲基氨基甲烷1.0mg·mL^-1和乙二胺四乙酸二钠钙0.1mg·mL^-12种辅料时,碘昔兰注射液最稳定。结论碘昔兰注射液应含辅料三羟甲基氨基甲烷1.0mg·mL^-1和乙二胺四乙酸二钠钙0.1mg·mL^-1。     

Effect of Carrier Excipient and Processing on Stability of Indorenate Hydrochloride/Excipient Mixtures

The purpose of this investigation was to determine the solid-state chemical stability of a model drug, indorenate hydrochloride, as a function of carrier excipient and mixing process. Physical mixtures and granules were prepared by tumble mixing and alcoholic granulation with and without binder. Stability of the mixtures was estimated using differential scanning calorimetry and isothermal degradation studies at 40, 50, and 60°C. Average first-order degradation constants at 25°C, extrapolated from isothermal studies, were much lower for indorenate hydrochloride after tumbling mixing with microcrystalline cellulose (3.45 × 10^{? 5} day^{? 1}) than those obtained after tumbling mixing with lactose (112.0 × 10^{? 5} day^{? 1}). Distribution of the drug on the excipient's surface, through granulation with and without Povidone, increased the average drug degradation rates in granules with microcrystalline cellulose (36.2 × 10^{? 5} day^{? 1}) as well as in granules with lactose (326 × 10^{? 5} day^{? 1}). Partially amorphous lactose (spray-dried lactose) showed higher average degradation rates (310.5 × 10^{? 5} day^{? 1}) than crystalline lactose (199.3 × 10^{? 5} day^{? 1}). It appears that the amorphous portion of the drug as well as that of reacting excipients play a major role in affecting the reaction rate. The calorimetric studies showed a strong solid–solid interaction between indorenate hydrochloride and lactose, suggesting chemical incompatibility. This strong solid–solid interaction was characterized by disappearance of typical transition peaks of lactose at temperatures above 200°C and the development of new peaks at about 130–170°C. No major changes in transition peaks were observed in mixtures of microcrystalline cellulose and indorenate hydrochloride, suggesting chemical compatibility. Calorimetric results allow the prediction of the chemical incompatibility between indorenate hydrochloride and lactose observed in isothermal degradation studies.     

Synergistic Effects of Multiple Excipients on Controlling Viscosity of Concentrated Protein Dispersions

Viscosity control is essential for the manufacturing and delivery of concentrated therapeutic proteins. Limited availability of the precious protein-based drugs hinders the characterization and screening of the formulation conditions with new types or different combinations of excipients. In this work, a droplet-based microfluidic device with incorporated multiple particle tracking microrheology (MPT) is developed to quantify the effects of two excipients, arginine hydrochloride (ArgHCl) and caffeine, on the viscosity of concentrated bovine gamma globulin (BGG) dispersions at two different values of pH. The effectiveness of both ArgHCl and caffeine show dependence on the BGG concentration and solution pH. The data set with high compositional resolution provides useful information to guide formulation with multiple viscosity-reducing excipients and quantification appropriate to start elucidating the connection to protein-protein interaction mechanisms. Overall, this work has demonstrated that the developed microfluidic approach has the potential to effectively assess the impact of multiple excipients on the viscosity and provide data for computational methods to predict viscosity for high concentration protein formulations.     

The Effects of Excipients on Protein Aggregation During Agitation: An Interfacial Shear Rheology Study

We investigated the effects of excipients in solutions of keratinocyte growth factor 2 (KGF-2) on protein aggregation during agitation as well as on interfacial shear rheology at the air–water interface. Samples were incubated with or without agitation, and in the presence or absence of the excipients heparin, sucrose, or polysorbate 80 (PS80). The effect of excipients on the extent of protein aggregation was determined by UV–visible spectroscopy and micro-flow imaging. Interfacial shear rheology was used to detect the gelation time and strength of protein gels at the air-water interface. During incubation, protein particles of size > 1μm and insoluble aggregates formed faster for KGF-2 solutions subjected to agitation. Addition of either heparin or sucrose promoted protein aggregation during agitation. In contrast, PS80 substantially inhibited agitation-induced KGF-2 aggregation but facilitated protein particulate formation in quiescent solutions. The combination of PS80 and heparin or sucrose completely prevented protein aggregation during both nonagitated and agitated incubations. Interfacial rheological measurements showed that KGF-2 in buffer alone formed an interfacial gel within a few minutes. In the presence of heparin, KGF-2 interfacial gels formed too quickly for gelation time to be determined. KGF-2 formed gels in about 10 min in the presence of sucrose. The presence of PS80 in the formulation inhibited gelation of KGF-2. Furthermore, the interfacial gels formed by the protein in the absence of PS80 were reversible when PS80 was added to the samples after gelation. Therefore, there is a correspondence between formulations that exhibited interfacial gelation and formulations that exhibited agitation-induced aggregation. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2460–2470, 2013     

辅料对阿司匹林片剂稳定性的影响

通过加速试验考察了几种填充剂，pH调节剂以及润滑剂对阿司匹林片剂稳定性的影响，阿司匹林降解产物采用HPLC法测定。结果表明；预胶化淀粉是最适宜的填充剂，1%枸橼酸的加入可降低阿司匹林的水解等；润滑剂则宜选用1%的十二烷基硫酸钠。     

Impact of Excipient Interactions on Solid Dosage Form Stability

Drug-excipient interactions in solid dosage forms can affect drug product stability in physical aspects such as organoleptic changes and dissolution slowdown, or chemically by causing drug degradation. Recent research has allowed the distinction in chemical instability resulting from direct drug-excipient interactions and from drug interactions with excipient impurities. A review of chemical instability in solid dosage forms highlights common mechanistic themes applicable to multiple degradation pathways. These common themes include the role of water and microenvironmental pH. In addition, special aspects of solid-state reactions with excipients and/or excipient impurities add to the complexity in understanding and modeling reaction pathways. This paper discusses mechanistic basis of known drug-excipient interactions with case studies and provides an overview of common underlying themes. Recent developments in the understanding of degradation pathways further impact methodologies used in the pharmaceutical industry for prospective stability assessment. This paper discusses these emerging aspects in terms of limitations of drug-excipient compatibility studies, emerging paradigms in accelerated stability testing, and application of mathematical modeling for prediction of drug product stability.     

Influence of Fluorescent Labelling of Polystyrene Particles on Phagocytic Uptake,Surface Hydrophobicity,and Plasma Protein Adsorption

Purpose. To investigate the influence of fluorescent labelling of polystyrene particles on phagocytic uptake, surface hydrophobicity and protein adsorption. Methods. Phagocytic uptake was analysed using chemiluminescence. Hydrophobicity was quantified by adsorption measurements of a hydrophobic dye. Protein adsorption was evaluated by two-dimensional electrophoresis. Results. Commercially available fluorescently labelled particles showed marked differences when compared to unlabelled particles: phagocytic uptake and surface hydrophobicity of labelled particles were diminished. Also the plasma protein adsorption pattern was found to be different from the unlabelled particles: for example, the amount of fibrinogen adsorbed was strongly reduced on the labelled particles. On the other hand, some unknown proteins could be detected on the fluorescently marked particles. In contrast, plain polystyrene particles and labelled ones could be successfully synthesised by Paulke which did not show any considerable differences in phagocytic uptake, surface hydrophobicity and protein adsorption. Polysorbate 20 added as stabilizer to particle suspensions led to completely different behaviour of the particles: the particles showed altered protein adsorption patterns, dominated by immunoglobulins and especially by apolipoproteins. Furthermore, these particles were not phagocytized at all. Conclusions. Surface hydrophobicity and phagocytic uptake in vitro as well as the interactions with plasma proteins of commercially available polystyrene particles were strongly affected by fluorescent labelling. Particles synthesised by Paulke remained unchanged after labelling. The results show the importance of thorough surface characterization for using particles in test systems in vitro and in vivo.     

Effects of Excipients on the Chemical and Physical Stability of Glucagon during Freeze-Drying and Storage in Dried Formulations

Purpose

To evaluate the effects of several buffers and excipients on the stability of glucagon during freeze-drying and storage as dried powder formulations.

Methods

The chemical and physical stability of glucagon in freeze-dried solid formulations was evaluated by a variety of techniques including mass spectrometry (MS), reversed phase HPLC (RP-HPLC), size exclusion HPLC (SE-HPLC), infrared (IR) spectroscopy, differential scanning calorimetry (DSC) and turbidity.

Results

Similar to protein drugs, maintaining the solid amorphous phase by incorporating carbohydrates as well as addition of surfactant protected lyophilized glucagon from degradation during long-term storage. However, different from proteins, maintaining/stabilizing the secondary structure of glucagon was not a prerequisite for its stability.

Conclusions

The formulation lessons learned from studies of freeze-dried formulations of proteins can be applied successfully to development of stable formulations of glucagon. However, peptides may behave differently than proteins due to their small molecule size and less ordered structure.     

Effects of Excipients on Protein Conformation in Lyophilized Solids by Hydrogen/Deuterium Exchange Mass Spectrometry

Purpose Excipients are added to lyophilized protein drug formulations to protect the protein during processing and storage, but the mechanisms are poorly understood. Here, hydrogen/deuterium (H/D) exchange with mass spectrometry was used to assess protein conformation and excipient interactions in lyophilized solids. Methods Calmodulin (CaM, 17 kD) was co-lyophilized with carbohydrate excipients (sucrose, mannitol, trehalose, raffinose, dextran 5,000, dextran 12,000) or guanidine hydrochloride (negative control) and exposed to D₂O vapor at 33% RH and RT. Samples were then dissolved and analyzed by mass spectrometry (+ESI/MS). Peptic digestion provided additional, site-specific information on H/D exchange. Solids were further characterized by powder x-ray diffraction (PXRD), differential scanning calorimetry (DSC), infrared spectroscopy (FTIR) and water vapor sorption. Results Excipients protected CaM from H/D exchange, increasing in the order guanidine hydrochloride < no excipient, mannitol < dextran 5,000, dextran 12,000 < sucrose < raffinose < trehalose. Effects were exerted primarily in the protein’s α-helical segments. Conclusions The effects of carbohydrate excipients on protein conformation in lyophilized solids are not exhibited uniformly along the protein sequence, but instead are exerted in a site-specific manner. The results also demonstrate the utility of H/D exchange with ESI/MS for protein structure characterization in lyophilized samples.     

The Effect of Formulation Excipients on Protein Stability and Aerosol Performance of Spray-Dried Powders of a Recombinant Humanized Anti-IgE Monoclonal Antibody1

Purpose. To study the effect of trehalose, lactose, and mannitol on the biochemical stability and aerosol performance of spray-dried powders of an anti-IgE humanized monoclonal antibody. Methods. Protein aggregation of spray-dried powders stored at various temperature and relative humidity conditions was assayed by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein glycation was determined by isoelectric focusing and affinity chromatography. Crystallization was examined by X-ray powder diffraction. Aerosol performance was assessed as the fine particle fraction (FPF) of the powders blended with coarse carrier lactose, and was determined using a multiple stage liquid impinger. Results. Soluble protein aggregation consisting of non-covalent and disulfide-linked covalent dimers and trimers occurred during storage. Aggregate was minimized by formulation with trehalose at or above a molar ratio in the range of 300:1 to 500:1 (excipient:protein). However, the powders were excessively cohesive and unsuitable for aerosol administration. Lactose had a similar stabilizing effect, and the powders exhibited acceptable aerosol performance, but protein glycation was observed during storage. The addition of mannitol also reduced aggregation, while maintaining the FPF, but only up to a molar ratio of 200:1. Further increased mannitol resulted in crystallization, which had a detrimental effect on protein stability and aerosol performance. Conclusions. Protein stability was improved by formulation with carbohydrate. However, a balance must be achieved between the addition of enough stabilizer to improve protein biochemical stability without compromising blended powder aerosol performance.     

A Modern View of Excipient Effects on Bioequivalence: Case Study of Sorbitol

Purpose To examine the effect of common excipients such as sugars (sorbitol versus sucrose) on bioequivalence between pharmaceutical formulations, using ranitidine and metoprolol as model drugs.Methods Two single-dose, replicated, crossover studies were first conducted in healthy volunteers (N = 20 each) to compare the effect of 5 Gm of sorbitol and sucrose on bioequivalence of 150 mg ranitidine or 50 mg metoprolol in aqueous solution, followed by a single-dose, nonreplicated, crossover study (N = 24) to determine the threshold of sorbitol effect on bioequivalence of 150 mg ranitidine in solution.Results Ranitidine Cmax and AUC(0–∞) were decreased by ∼50% and 45%, respectively, in the presence of sorbitol versus sucrose. Similarly, sorbitol reduced metoprolol Cmax by 23% but had no significant effect on AUC(0–∞). An appreciable subject-by-formulation interaction was found for ranitidine Cmax and AUC(0–∞), as well as metoprolol Cmax. Sorbitol decreased the systemic exposure of ranitidine in a dose-dependent manner and affected bioequivalence at a level of 1.25 Gm or greater.Conclusions As exemplified by sorbitol, some common excipients have unexpected effect on bioavailability/bioequivalence, depending on the pharmacokinetic characteristics of the drug, as well as the type and amount of the excipient present in the formulation. More research is warranted to examine other ‘common’ excipients that may have unintended influence on bioavailability/bioequivalence.The opinions expressed in this article are those of the authors and do not necessarily represent the views or policies of the Food and Drug Administration.     

药用辅料二氧化硅生产工艺稳定性评价策略与分析

目的：基于同等重要的质量标准指标和功能性相关指标,提出生产工艺稳定性评价策略作为药用辅料质量控制的一种新方法。方法：首先测定了A、B厂家共14批次二氧化硅的质量标准指标（干燥失重、炽灼失重、酸碱度和含量）和功能性相关指标（引湿性、比表面积、粒度和粒度分布）。然后,通过生产工艺稳定性评价策略获得质量标准指标稳定性差值（PQ）和功能性相关指标稳定性差值 （PF）。结果：A厂家和B厂家二氧化硅的PQ值分别为31.1%和58.1%,PF值分别为15.6%和50.0%。结论：通过比较2个厂家的PQ值和PF值的大小,可以反映特定生产厂家对产品杂质控制效果的好坏和对功能性指标控制与否以及控制要求的差异,从而全面反映特定企业对其产品生产工艺稳定性的控制。PQ 值和PF值为监测二氧化硅生产工艺稳定性提供了一种全面有效的方法,可为其他药用辅料生产工艺稳定性监测提供参考。     

Determination of Paclitaxel Solubility and Stability in the Presence of Injectable Excipients

Pharmaceutical Chemistry Journal - Due to poor aqueous solubility of paclitaxel, cremophor is one of the excipients used to improve solubility in Taxol while it is responsible for a number of...     

盐酸林可霉素注射液辅料对产品质量的影响探讨

目的：探讨辅料对盐酸林可霉素注射液产品质量的影响。方法：对50家生产企业223批次国家药品评价抽验样品进行检验,并对辅料与表征产品质量的多个指标进行相关性统计分析。结果：添加苯甲醇不利于药品的稳定,EDTA-二钠、亚硫酸氢钠、焦亚硫酸钠、枸橼酸钠有利于药品的稳定,醋酸钠、磷酸二氢钠、碳酸氢钠对药品质量无显著影响。结论：部分不合理处方应在科学评价的基础上,进行修改。     

Influence of Surface Properties at Biodegradable Microsphere Surfaces: Effects on Plasma Protein Adsorption and Phagocytosis

Purpose. The objective of this work was to determine plasma protein adsorption and macrophage phagocytosis of biodegradable polyanhydride, polylactic acid and polylactic-co-glycolic acid microspheres prepared by both spray-drying and solvent evaporation techniques. Methods. Microspheres were characterized by scanning electron microscopy (SEM), confocal laser microscopy, particle size distribution and zeta () potential determination. Plasma protein adsorption onto the microspheres was determined using a fluoroaldehyde reagent. Phagocytosis was evaluated by incubating microspheres containing the angiotensin II antagonist, L-158,809, with the macrophages in the presence or absence of the phagocytosis inhibitor cythochalasin D. The extent of phagocytosis was established by fluorescence determination of L-158,809 and by optical microscopy. The effect of amphiphilic poly(ethylene glycol) (PEG) derivatives on phagocytosis was determined using PEG-distearate incorporated into the microspheres. Results. The average diameter of the microspheres, which depended on the polymer and the initial formulation, ranged from 0.9 to 3.2 micrometers. potential studies showed strong negative values irrespective of the polymer used for the spray-dried formulations. The potential was masked by the incorporation of PEG 400- or PEG 1,400-distearate in the formulation. Confocal laser microscopy showed a homogenous dispersion of PEG (measured as PEG-fluorescein) in the microspheres. Protein adsorption was not observed for any of the microsphere formulations following incubation with bovine serum. Incubation of microspheres with murine macrophages showed that PEG-distearate inhibited phagocytosis at appropriate levels (0,1% w/w). Higher levels >1% w/w of PEG-distearate) resulted in enhanced association with macrophages, despite the presence of the phagocytosis inhibitor cytochalasin D, indicating fusion between the microspheres and the plasma membrane. Conclusions. These results demonstrate that spray-dried PEG-containing microspheres can be manufactured and that an appropriate concentration of this excipient in microspheres results in decreased phagocytosis.     

Inhibitors of the FcRn:IgG Protein–Protein Interaction

The neonatal Fc receptor, FcRn, is responsible for controlling the half-life of IgG antibodies. As a result, inhibitors of FcRn have been investigated as a possible way to modulate IgG half-lives. Such inhibitors could have possible applications in reducing autoantibody levels in autoimmune disease states. To date, monoclonal antibodies, engineered Fc domains, and short peptides have been reported to inhibit FcRn function and modulate IgG half-lives in vivo.     

不同辅料对硝苯地平缓释片光稳定性影响研究

目的 考察硝苯地平缓释片中原料药与辅料的相容性,为更好地设计处方,控制和提高质量提供依据和信息。方法 采用中国药典2015年版二部硝苯地平原料药项下有关物质检查方法,考察有关物质变化,作为不同辅料对硝苯地平缓释片光稳定性的影响指标。结果 在光照条件下,硝苯地平易产生光降解杂质2,且微晶纤维素和硬脂酸镁会加速杂质2的产生。结论 在满足相关制剂要求的条件下,建议尽量少加或不加微晶纤维素和硬脂酸镁。     

Effects of Various Pharmaceutical Excipients on the Intestinal Transport and Absorption of Sulfasalazine,a Typical Substrate of Breast Cancer Resistance Protein Transporter

Breast cancer resistance protein (BCRP) transporter is an efflux transporter that utilizes energy from adenosine triphosphate hydrolysis to push its substrates, regardless of the concentration gradient. Its presence on the apical membrane of the intestinal mucosa is a major obstacle for the intestinal absorption of its substrates. In this study, we examined the effects of various pharmaceutical excipients on the intestinal transport and absorption of sulfasalazine, a BCRP substrate. Four excipients, including 0.05% and 0.075% BL-9EX, 0.01% and 0.05% Brij 97, 0.075% Labrasol, and 0.05% and 0.1% Tween 20 decreased the secretory transport of sulfasalazine in an in vitro diffusion chamber. Further investigation in an in situ closed loop experiment in rats showed that 0.05% and 0.1% BL-9EX and 0.1% Brij 97 effectively enhanced the intestinal absorption of sulfasalazine while maintaining minimal toxicity to the intestinal mucosa. However, 0.1% Brij 97 also increased the intestinal absorption of 5(6)-carboxyfluorescein, a paracellular marker compound. These findings suggest that BL-9EX might effectively inhibit the BCRP-mediated efflux of sulfasalazine in vivo, indicating that BL-9EX could improve the intestinal absorption of sulfasalazine and other BCRP substrates.