Schwendeman S P, Costantino H R, Gupta R K, Siber G R, Klibanov A M, Langer R
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge 02139, USA.
Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11234-8. doi: 10.1073/pnas.92.24.11234.
The progress toward single-dose vaccines has been limited by the poor solid-state stability of vaccine antigens within controlled-release polymers, such as poly(lactide-co-glycolide). For example, herein we report that lyophilized tetanus toxoid aggregates during incubation at 37 degrees C and elevated humidity--i.e., conditions relevant to its release from such systems. The mechanism and extent of this aggregation are dependent on the moisture level in the solid protein, with maximum aggregation observed at intermediate moisture contents. The main aggregation pathway is consistent with formaldehyde-mediated cross-linking, where reactive electrophiles created and stored in the vaccine upon formalinization (exposure to formaldehyde during vaccine preparation) react with nucleophiles of a second vaccine molecule to form intermolecular cross-links. This process is inhibited by the following: (i) succinylating the vaccine to block reactive amino groups; (ii) treating the vaccine with sodium cyanoborohydride, which presumably reduces Schiff bases and some other electrophiles created upon formalinization; and (iii) addition of low-molecular-weight excipients, particularly sorbitol. The moisture-induced aggregation of another formalinized vaccine, diphtheria toxoid, is also retarded by succinylation, suggesting the generality of this mechanism for formalinized vaccines. Hence, mechanistic stability studies of the type described herein may be important for the development of effective single-dose vaccines.
在向单剂量疫苗发展的过程中，疫苗抗原在控释聚合物（如聚乳酸 - 乙醇酸共聚物）中的固态稳定性较差，这限制了其发展。例如，在此我们报告，冻干的破伤风类毒素在37℃和高湿度条件下孵育时会发生聚集，即与其从此类系统中释放的相关条件下会发生聚集。这种聚集的机制和程度取决于固体蛋白质中的水分含量，在中等水分含量时观察到最大聚集。主要的聚集途径与甲醛介导的交联一致，即疫苗在福尔马林化（疫苗制备过程中暴露于甲醛）时产生并储存的活性亲电试剂与第二个疫苗分子的亲核试剂反应形成分子间交联。以下方法可抑制此过程：（i）使疫苗琥珀酰化以阻断反应性氨基；（ii）用氰基硼氢化钠处理疫苗，据推测这会还原福尔马林化时产生的席夫碱和其他一些亲电试剂；（iii）添加低分子量赋形剂，特别是山梨醇。琥珀酰化也可抑制另一种福尔马林化疫苗白喉类毒素的水分诱导聚集，这表明这种机制对福尔马林化疫苗具有普遍性。因此，本文所述类型的机理稳定性研究对于开发有效的单剂量疫苗可能很重要。
