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首页> 外文期刊>Journal of pharmaceutical sciences. >Evaluation of chemical degradation of a trivalent recombinant protein vaccine against botulinum neurotoxin by LysC peptide mapping and MALDI-TOF mass spectrometry.
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Evaluation of chemical degradation of a trivalent recombinant protein vaccine against botulinum neurotoxin by LysC peptide mapping and MALDI-TOF mass spectrometry.

机译：通过LysC肽图分析和MALDI-TOF质谱评估抗肉毒杆菌神经毒素的三价重组蛋白疫苗的化学降解。

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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 et al., in press, J Pharm Sci), we characterized the effects of binding to adjuvant on the conformation and thermodynamic stability of three antigen variants for botulinum vaccines: rBoNTA(H(c)), rBoNTB(H(c)), and rBoNTE(H(c)). 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(H(c)) as well as a trivalent rBoNT(H(c)) vaccine formulation. This method provided high sequence coverage for the proteins in part due to the implementation of a postdigestion elution fractionation method during sample preparation, and was also successfully utilized to evaluate the chemical integrity of adjuvant-bound rBoNT(H(c)) protein antigens. We found that all three of the rBoNT(H(c)) 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.
机译：利用重组蛋白抗原的疫苗通常需要佐剂来增强受体的免疫反应。但是，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。在我们的伴侣论文中（Vessely等人，印刷中，J Pharm Sci），我们表征了结合佐剂对三种肉毒杆菌疫苗抗原变体构象和热力学稳定性的影响：rBoNTA（H（c）），rBoNTB H（c））和rBoNTE（H（c））。在当前的研究中，我们评估了在水悬浮液中长期存储期间，结合佐剂（Alhydrogel，氢氧化铝）对这些抗原的化学稳定性的影响。我们开发了将LysC肽图谱与MALDI-TOF质谱结合使用的方法。针对用于rBoNTE（H（c））疫苗制剂以及三价rBoNT（H（c））疫苗制剂的蛋白质开发了肽图。该方法为蛋白质提供了较高的序列覆盖率，部分原因是在样品制备过程中实施了消化后洗脱分级分离方法，并且还成功地用于评估佐剂结合的rBoNT（H（c））蛋白抗原的化学完整性。我们发现，所有三个rBoNT（H（c））蛋白都易于通过氧化和脱酰胺作用降解。在许多情况下，与未与佐剂结合的对照样品中的蛋白质相比，这种与佐剂结合的蛋白质制剂的反应更早发生。另外，在佐剂结合的蛋白质制剂中发现了一些化学修饰，但在未结合的溶液对照中未检测到。我们的研究表明与铝基佐剂的结合会影响在水悬浮液中长期存储期间蛋白质的化学稳定性和/或化学降解途径。此外，我们开发的方法应可广泛用于评估佐剂结合的重组蛋白抗原的化学稳定性。

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