Institute of Animal Cell Technology and Systems Biology, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
BOKU Core Facility Mass Spectrometry, University of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria.
Int J Mol Sci. 2023 Jun 30;24(13):10891. doi: 10.3390/ijms241310891.
CD19 is an essential protein in personalized CD19-targeting chimeric antigen receptor (CAR)-T cell-based cancer immunotherapies and CAR-T cell functionality evaluation. However, the recombinant expression of this "difficult to-express" (DTE) protein is challenging, and therefore, commercial access to the protein is limited. We have previously described the successful stable expression of our soluble CD19-AD2 fusion protein of the CD19 extracellular part fused with human serum albumin domain 2 (AD2) in CHO-K1 cells. The function, stability, and secretion rate of DTE proteins can be improved by culture conditions, such as reduced temperature and a shorter residence time. Moreover, glycosylation, as one of the most important post-translational modifications, represents a critical quality attribute potentially affecting CAR-T cell effector function and thus impacting therapy's success. In this study, we increased the production rate of CD19-AD2 by 3.5-fold through applying hypothermic culture conditions. We efficiently improved the purification of our his-tagged CD19-AD2 fusion protein via a Ni-NTA-based affinity column using a stepwise increase in the imidazole concentration. The binding affinity to commercially available anti-CD19 antibodies was evaluated via Bio-Layer Interferometry (BLI). Furthermore, we revealed glycosylation patterns via Electrospray Ionization Mass Spectrometry (ESI-MS), and five highly sialylated and multi-antennary N-glycosylation sites were identified. In summary, we optimized the CD19-AD2 production and purification process and were the first to characterize five highly complex N-glycosylation sites.
CD19 是个性化 CD19 靶向嵌合抗原受体（CAR）-T 细胞癌症免疫疗法和 CAR-T 细胞功能评估中必不可少的蛋白质。然而，这种“难表达”（DTE）蛋白的重组表达具有挑战性，因此，商业上获得该蛋白的途径有限。我们之前已经描述了我们的可溶性 CD19-AD2 融合蛋白在 CHO-K1 细胞中的稳定表达，该融合蛋白由 CD19 细胞外部分与人血清白蛋白结构域 2（AD2）融合而成。通过降低温度和缩短停留时间等培养条件，可以提高 DTE 蛋白的功能、稳定性和分泌率。此外，糖基化作为最重要的翻译后修饰之一，是一个关键的质量属性，可能会影响 CAR-T 细胞的效应功能，从而影响治疗的成功。在这项研究中，我们通过应用低温培养条件将 CD19-AD2 的产率提高了 3.5 倍。我们通过逐步增加咪唑浓度，有效地提高了 Ni-NTA 亲和柱对我们的 His 标记的 CD19-AD2 融合蛋白的纯化效率。通过生物层干涉（BLI）评估了与市售抗 CD19 抗体的结合亲和力。此外，我们通过电喷雾电离质谱（ESI-MS）揭示了糖基化模式，并鉴定了五个高度唾液酸化和多天线 N-糖基化位点。总之，我们优化了 CD19-AD2 的生产和纯化工艺，并首次对五个高度复杂的 N-糖基化位点进行了表征。
