論文掲載：Sanofi社、糖ペプチドライブラリーを用いたSf9由来SARS-CoV-2スパイクタンパク質組み換えワクチン候補の糖ペプチド特性解析の高速化

Rapid Commun Mass Spectrom
January 25, 2023

Sanofiの研究者はGenedataとのコラボレーションを通じ、糖ペプチドライブラリーを用いた自動質量分析アプローチによって、大きな糖タンパク質の部位特異的な糖鎖占有率データの作成を高速化しました。本論文では、COVID-19ワクチン抗原候補として開発中のSf9昆虫細胞由来組み換えスパイクタンパク質におけるN-結合グリコシル化パターンを報告しています。

In this study, reaseachers from Sanofi report the N-glycosylation pattern of a Sf9 insect cell derived recombinant spike proteins being developed as candidate vaccine antigens for SARS-CoV-2 (COVID-19).

Rationale

The method has been optimised to produce peptides with single, isolated glycosylation sites using multiple protease digests. The development and use of glycopeptide libraries from previous developmental phases allowed for faster analysis than processing data sets from individual batches from first principles.

Methods

Purified spike proteins were reduced, alkylated, and digested with proteolytic enzymes. Three different protease digests were utilised to generate peptides with isolated glycosylation sites. The glycopeptides were then analysed using a Waters Q-TOF while using a data dependent acquisition (DDA) mass spectrometry experiment. Glycopeptide mapping data processing and glycan classification was performed using Genedata Expressionist via a specialised workflow that used libraries of previously detected glycopeptides to greatly reduce processing time.

Results

Two different spike proteins from 6 manufacturers were analysed. There was a strong similarity at each site across batches and manufacturers. The majority of the glycans present were of the truncated class, although at sites N61, N234, and N717/714 high mannose structures were dominant and at N1173/1170 aglycosylation was dominant for both variant proteins. A comparison was performed on a commercially available spike protein and our results were found to be similar to earlier reports.

Conclusions

Our data clearly shows that the overall glycosylation pattern of both spike protein variants was highly similar from batch-to-batch, and between materials produced at different manufacturing facilities. The use of our glycopeptide libraries greatly expedited the generation of site-specific glycan occupancy data for a large glycoprotein. We compared our method with previously obtained data from a commercially available insect cell derived spike protein and the results were comparable to published findings.