Biomolecular Formulation and Characterisation Sciences, UCB Pharma, Braine l'Alleud, Belgium.
Biomolecular Formulation and Characterisation Sciences, UCB Pharma, Braine l'Alleud, Belgium.
Eur J Pharm Biopharm. 2020 Nov;156:131-142. doi: 10.1016/j.ejpb.2020.08.019. Epub 2020 Aug 31.
To increase their stability, therapeutic (or monoclonal) antibodies (mAbs) are often formulated as solids by using a variety of drying techniques, e.g. freeze-drying, spray-drying, or spray freeze-drying. The addition of excipients is required to preserve stability of the protein during the drying process and subsequent storage of the resulting solid form. The addition of low molecular weight excipients, such as amino acids, to sugar based spray- and freeze-dried formulations has been suggested to improve the storage stability of proteins in the dried state. In this study sugars (sucrose, trehalose), amino acids (Gly, Ala, Pro, Ser, Val, Leu, Ile, Gln, His, Lys, Arg, Phe, Trp) and combinations thereof were investigated for their stabilizing effect during spray-drying and subsequent storage and for their reconstitution time reducing effect. Two IgG4 mAbs were used as model antibodies. From an initial screening study, basic and small neutral amino acids, in combination with a sugar, such as sucrose or trehalose, showed reconstitution time reducing and stabilizing properties. Arg in particular displayed excellent reconstitution and stability enhancing properties. Moreover, Arg was the only amino acid providing stabilizing properties comparable to sucrose or trehalose. Previous work by the authors described a statistically substantiated comparison between the three basic amino acids in a sugar containing formulation, albeit limited to a single concentration level [5]. Therefore, a follow-up design of experiments (DoE) study was performed to determine the optimum trehalose/amino acid content required for an optimal protein stability and reconstitution time and to compare the effects of two basic amino acids, Lys and Arg, to those of two neutral amino acids, Gly and Pro. The conducted DoE covered a wide range of trehalose (30-120 mM) and amino acid (50-150 mM) concentrations. The concentration of trehalose was found to be the main contributor to a reduction in reconstitution time and an increase in stability. Here we show that the addition of amino acids such as Gly, Pro, and Lys does not improve stability nor does it reduce the reconstitution time. Of the tested amino acids, only Arg showed a marked reduction in reconstitution time and improvement in stability compared to a trehalose. Moreover, the properties displayed by Arg could justify its application as the main stabilizer in spray-dried mAb formulations, eliminating the need for a sugar matrix altogether. But the weight ratio of stabilizer to protein was found the factor exerting the strongest overall influence on the formulation's reconstitution time and stability. More specifically, sufficient physical stability and an acceptable reconstitution time could be obtained with a protein to stabilizer weight ratio of at least 1:1.
为了提高稳定性，治疗性（或单克隆）抗体（mAbs）通常通过使用各种干燥技术（例如冷冻干燥、喷雾干燥或喷雾冷冻干燥）制成固体。需要添加赋形剂以在干燥过程中和随后储存所得固体形式期间保持蛋白质的稳定性。向基于糖的喷雾干燥和冷冻干燥制剂中添加低分子量赋形剂，如氨基酸，已被建议可以提高蛋白质在干燥状态下的储存稳定性。在这项研究中，研究了糖（蔗糖、海藻糖）、氨基酸（甘氨酸、丙氨酸、脯氨酸、丝氨酸、缬氨酸、亮氨酸、异亮氨酸、谷氨酰胺、组氨酸、赖氨酸、精氨酸、苯丙氨酸、色氨酸）及其组合在喷雾干燥和随后的储存过程中的稳定作用，以及它们的复溶时间缩短效果。使用两种 IgG4 mAbs 作为模型抗体。通过初步筛选研究，碱性和小中性氨基酸与糖（如蔗糖或海藻糖）结合显示出复溶时间缩短和稳定的特性。特别是 Arg 显示出极好的复溶和稳定增强特性。此外，Arg 是唯一提供与蔗糖或海藻糖相当的稳定特性的氨基酸。作者之前的工作描述了在含糖制剂中三种碱性氨基酸之间的统计学上合理的比较，尽管仅限于单一浓度水平[5]。因此，进行了后续的实验设计（DoE）研究，以确定最佳海藻糖/氨基酸含量，以获得最佳的蛋白质稳定性和复溶时间，并比较两种碱性氨基酸，赖氨酸和精氨酸，与两种中性氨基酸，甘氨酸和脯氨酸的效果。进行的 DoE 涵盖了广泛的海藻糖（30-120 mM）和氨基酸（50-150 mM）浓度。发现海藻糖浓度是降低复溶时间和增加稳定性的主要因素。在这里，我们表明添加甘氨酸、脯氨酸和赖氨酸等氨基酸不会提高稳定性，也不会缩短复溶时间。在所测试的氨基酸中，只有精氨酸与海藻糖相比，复溶时间明显缩短，稳定性提高。此外，与海藻糖相比，精氨酸所表现出的特性可以证明其在喷雾干燥 mAb 制剂中作为主要稳定剂的应用是合理的，从而完全消除了对糖基质的需求。但是，稳定剂与蛋白质的重量比被发现是对制剂复溶时间和稳定性产生最强总体影响的因素。更具体地说，通过使用至少 1:1 的蛋白质与稳定剂的重量比，可以获得足够的物理稳定性和可接受的复溶时间。
