Leicester School of Pharmacy, Faculty of Health and Life Sciences, De Montfort University, Leicester, UK.
School of Pharmacy, University of Reading, Reading, UK.
AAPS PharmSciTech. 2018 Oct;19(7):2934-2947. doi: 10.1208/s12249-018-1114-0. Epub 2018 Jul 6.
The current paper continues our study on the ability of L-arginine to prevent/reduce the aggregation of proteins that results from the various stresses during the lyophilisation and/or storage of lyophilized protein-based products. The first part of our study, i.e. formulation development, was devoted to the rational design and optimization of an L-arginine containing lyophilized formulation which can resist the natural tendency of L-arginine to absorb atmosphere moisture. Mannitol and trehalose were chosen among other excipients to be included in the protein-based formulation, as mannitol in a combination with L-arginine has been shown to reduce moisture sorption while trehalose provides a degree of lyoprotection. In the present study, a number of formulations, which comprised bovine serum albumin (BSA) with and without L-arginine, and with five different ratios of trehalose-to-mannitol (from 30:70 to 80:20) were lyophilised and assessed. The internal structures and the moisture sorption/retention of the lyophilized formulations were characterised. To study the effect of L-arginine on BSA solid-phase stability, the lyophilized powder was exposed to accelerated storage conditions (high moisture (75% RH) and temperature (22 or 45 °C)) for up to 24 h. The lyophilized BSA formulations were then reconstituted and solution-state protein aggregation assessed by turbidimetry at 360 nm and fluorescence spectroscopy using the thioflavin T assay. It was demonstrated that L-arginine can be used in protein-based freeze-dried formulations to significantly reduce the aggregation of protein during the manufacturing, storage and subsequent reconstitution. The results also revealed the importance of a sufficient amount of mannitol in the arginine-containing formulations.
本文继续研究 L-精氨酸防止/减少蛋白质在冷冻干燥和/或冷冻干燥蛋白产品储存过程中各种应激引起的聚集的能力。我们研究的第一部分，即配方开发，致力于合理设计和优化含有 L-精氨酸的冷冻干燥配方，该配方可以抵抗 L-精氨酸吸收大气水分的自然趋势。甘露醇和海藻糖等赋形剂被选入蛋白基配方中，因为 L-精氨酸与甘露醇的组合已被证明可以减少水分吸收，而海藻糖提供一定程度的冷冻保护。在本研究中，制备了包含牛血清白蛋白（BSA）和不含 L-精氨酸的几种配方，以及含有五种不同海藻糖与甘露醇比例（30:70 至 80:20）的配方，并对其进行了冷冻干燥和评估。对冷冻干燥配方的内部结构和水分吸附/保留进行了表征。为了研究 L-精氨酸对 BSA 固相稳定性的影响，将冷冻干燥的粉末暴露于加速储存条件（高湿度（75%RH）和温度（22 或 45°C））下长达 24 小时。然后将冷冻干燥的 BSA 配方重新配制，并通过 360nm 浊度法和使用硫代黄素 T 测定法的荧光光谱法评估溶液状态下蛋白质的聚集。结果表明，L-精氨酸可用于基于蛋白质的冷冻干燥配方中，以显著减少蛋白质在制造、储存和随后再配制过程中的聚集。结果还揭示了含精氨酸配方中甘露醇含量充足的重要性。
