Production of glycosylated human anti-Hepatitis B monoclonal antibodies in the microalgae Phaeodactylum tricornutum

Monoclonal antibodies (mAbs) represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fuelled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N- glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing homogenous recombinant proteins bearing high mannose-type N-glycans.

单克隆抗体（monoclonal antibodies, mAbs）实为生物制药领域的核心品类。此类抗体通过重组技术，以活细胞作为生物工厂进行生产。在当前可用的各类蛋白表达系统中，微藻是一类新兴替代方案，具备多项生物技术优势。事实上，微藻被归类为公认安全（generally recognized as safe, GRAS）生物体，可在生物反应器中高效培养，生长速率与中国仓鼠卵巢（CHO）细胞相当。此外，微藻为光合自养型生物，其蛋白质表达可通过光合作用供能，因此生产成本低廉。然而，在实现藻类源生物制药的工业化生产前，仍有诸多问题亟待解决。其中，蛋白质异质性与蛋白质翻译后修饰的相关问题亟需评估。尤为关键的是，分泌型重组蛋白所获得的N-糖基化修饰备受关注——因包括单克隆抗体在内的多数生物制药均为N-糖基化修饰，且糖基化已被广泛认定为其关键质量属性之一。本研究针对在硅藻三角褐指藻（Phaeodactylum tricornutum）中表达的首款藻类源重组单克隆抗体的质量展开评估，重点对其C端与N端末端、信号肽切割情况，以及包括N-糖基化宏观与微观异质性在内的各类翻译后修饰进行表征。本研究深化了人们对硅藻细胞生物学，尤其是蛋白质分泌与胞内运输机制的认知。总体而言，本研究进一步确立了三角褐指藻作为生物制药生产新兴宿主的定位，并证实该藻株可用于生产携带高甘露糖型N-聚糖的均一重组蛋白。