Department of Pharmaceutical Chemistry, Vaccine Analytics and Formulation Center, University of Kansas, Lawrence, Kansas 66047.
The Center for Vaccine Innovation and Access, PATH, Seattle, Washington 98121.
J Pharm Sci. 2020 Jan;109(1):476-487. doi: 10.1016/j.xphs.2019.10.004. Epub 2019 Oct 4.
A nonreplicating rotavirus vaccine (NRRV) containing 3 recombinant fusion proteins adsorbed to aluminum adjuvant (Alhydrogel [AH]) is currently in clinical trials. The compatibility and stability of monovalent NRRV antigen with key components of a multidose vaccine formulation were examined using physicochemical and immunochemical methods. The extent and strength of antigen-adjuvant binding were diminished by increasing phosphate concentration, and acceptable levels were identified along with alternate buffering agents. Addition of the preservative thimerosal destabilized AH-adsorbed P2-VP8-P[8] as measured by differential scanning calorimetry. Over 3 months at 4°C, AH-adsorbed P2-VP8-P[8] was stable, whereas at 25°C and 37°C, instability was observed which was greatly accelerated by thimerosal addition. Loss of antibody binding (enzyme-linked immunosorbent assay) correlated with loss of structural integrity (differential scanning calorimetry, fluorescence spectroscopy) with concomitant nonnative disulfide bond formation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Asn deamidation (liquid chromatography -mass spectrometry peptide mapping). An alternative preservative (2-phenoxyethanol) showed similar antigen destabilization. Due to limited availability, only key assays were performed with monovalent P2-VP8-P[4] and P2-VP8-P[6] AH-adsorbed antigens, and varying levels of preservative incompatibility were observed. In summary, monovalent AH-adsorbed NRRV antigens stored at 4°C showed good stability without preservatives; however, future formulation development efforts are required to prepare a stable, preservative-containing, multidose NRRV formulation.
一种含有 3 种重组融合蛋白的非复制型轮状病毒疫苗（NRRV），与铝佐剂（Alhydrogel [AH]）结合，目前正在临床试验中。使用物理化学和免疫化学方法，研究单价 NRRV 抗原与多剂量疫苗配方关键成分的兼容性和稳定性。通过增加磷酸盐浓度，降低了抗原-佐剂结合的程度和强度，并确定了可接受的水平和替代缓冲剂。添加防腐剂硫柳汞通过差示扫描量热法使 AH 吸附的 P2-VP8-P[8]不稳定。在 4°C 下超过 3 个月，AH 吸附的 P2-VP8-P[8]稳定，而在 25°C 和 37°C 下，观察到不稳定，添加硫柳汞大大加速了不稳定。抗体结合（酶联免疫吸附测定）的丧失与结构完整性的丧失（差示扫描量热法、荧光光谱法）相关，同时伴随着非天然二硫键形成（十二烷基硫酸钠-聚丙烯酰胺凝胶电泳）和 Asn 脱酰胺（液相色谱 -质谱肽图谱）。替代防腐剂（2-苯氧乙醇）也显示出类似的抗原不稳定。由于供应有限，仅对单价 P2-VP8-P[4]和 P2-VP8-P[6] AH 吸附抗原进行了关键检测，观察到不同水平的防腐剂不兼容。总之，在 4°C 下储存的单价 AH 吸附 NRRV 抗原在没有防腐剂的情况下表现出良好的稳定性；然而，需要进一步的制剂开发工作来制备稳定的、含防腐剂的、多剂量 NRRV 制剂。
