Evaluation of chemical degradation of a trivalent recombinant protein vaccine against botulinum neurotoxin by LysC peptide mapping and MALDI-TOF mass spectrometry.
Evaluation of chemical degradation of a trivalent recombinant protein vaccine against botulinum neurotoxin by LysC peptide mapping and MALDI-TOF mass spectrometry.
复制标题
DOI：
10.1002/jps.21543
复制
发表时间：
2009-09
影响因子：
3.8
通讯作者：
Carpenter, John F.
中科院分区：
文献类型：
--
作者：
Estey, Tia;Vessely, Christina;Randolph, Theodore W.;Henderson, Ian;Braun, Latoya Jones;Nayar, Rajiv;Carpenter, John F.
关键词：
利用重组蛋白抗原的疫苗通常需要佐剂来增强接受者中的免疫应答。然而，抗原与佐剂结合对蛋白质的短期和长期稳定性的影响仍然不清楚。在我们的配套论文（Vessely C，et al.，2007），我们表征了与佐剂结合对肉毒杆菌疫苗的三种抗原变体的构象和热力学稳定性的影响：rBoNTA（Hc）、rBoNTB（Hc）和rBoNTE（Hc）。在本研究中，我们评估了与佐剂（Alhydrogel™，氢氧化铝）结合对这些抗原在水性悬浮液中长期储存期间的化学稳定性的影响。我们开发了采用LysC肽图谱结合MALDI-TOF质谱的方法。针对rBoNTE（Hc）疫苗制剂以及三价rBoNT（Hc）疫苗制剂的蛋白质开发肽图谱。这种方法提供了高的序列覆盖率的蛋白质，部分原因是在样品制备过程中的消化后洗脱分级方法的实施，也被成功地用于评估化学完整性的澄清剂结合的rBoNT（HC）蛋白抗原。我们发现，所有三种rBoNT（Hc）蛋白易于通过氧化和脱酰胺降解。在许多情况下，当与未与佐剂结合的对照样品中的蛋白质相比时，与佐剂结合的蛋白质制剂的这种反应发生得更早。此外，在澄清剂结合蛋白制剂中发现了一些化学修饰，但在未结合溶液对照中未检测到。我们的研究表明，与铝基佐剂的结合可以影响蛋白质在水悬浮液中长期储存期间的化学稳定性和/或化学降解途径。此外，我们开发的方法应该是广泛适用于评估化学稳定性的促凝剂结合的重组蛋白抗原。
Vaccines utilizing recombinant protein antigens typically require an adjuvant to enhance immune response in the recipients. However, the consequences of antigen binding to adjuvant on both the short- and long-term stability of the protein remain poorly defined. In our companion paper (Vessely C, et al., 2007), we characterized the effects of binding to adjuvant on the conformation and thermodynamic stability of three antigen variants for botulinum vaccines: rBoNTA(Hc), rBoNTB(Hc), and rBoNTE(Hc). In the current study we evaluated the effect of binding to adjuvant (Alhydrogel™, aluminum hydroxide) on chemical stability of these antigens during long-term storage in aqueous suspension. We developed methods that employ LysC peptide mapping in conjunction with MALDI-TOF mass spectrometry. Peptide maps were developed for the proteins for a vaccine formulation of rBoNTE(Hc), as well as a trivalent rBoNT(Hc) vaccine formulation. This method provided high sequence coverage for the proteins in part due to the implementation of a post-digestion elution fractionation method during sample preparation, and was also successfully utilized to evaluate the chemical integrity of adjuvant-bound rBoNT(Hc) protein antigens. We found that all three of the rBoNT(Hc) proteins were susceptible to degradation via both oxidation and deamidation. In many cases, such reactions occurred earlier with the adjuvant-bound protein formulations when compared to the proteins in control samples that were not bound to adjuvant. Additionally, some chemical modifications were found in the adjuvant-bound protein formulations but were not detected in the unbound solution controls. Our studies indicate that binding to aluminum-based adjuvants can impact the chemical stability and/or the chemical degradation pathways of protein during long-term storage in aqueous suspension. Furthermore, the methods we developed should be widely useful for assessing chemical stability of adjuvant-bound recombinant protein antigens.
登录
查看更多内容
DOI：
10.1073/pnas.0609713104
发表时间：
2007-01-02
影响因子：
11.1
作者：
Rummel, Andreas;Eichner, Timo;Binz, Thomas
通讯作者：
Binz, Thomas
影响因子：
4.8
作者：
Flaugh, Shannon L.;Mills, Ishara A.;King, Jonathan
通讯作者：
King, Jonathan
DOI：
10.1002/path.1700340214
发表时间：
1931-01-01
期刊：
JOURNAL OF PATHOLOGY AND BACTERIOLOGY
影响因子：
--
作者：
Glenny, AT;Buttle, GAH;Stevens, MF
通讯作者：
Stevens, MF
影响因子：
3.1
作者：
ELDRIDGE, JH;STAAS, JK;GILLEY, RM
通讯作者：
GILLEY, RM
影响因子：
2.8
作者：
Rosenberg, JS;Middlebrook, JL;Atassi, MZ
通讯作者：
Atassi, MZ