Production of glycoprotein B from human cytomegalovirus in glycoengineered Nicotiana tabacum Bright Yellow-2 suspension cells

The human cytomegalovirus (HCMV) is a widespread virus and is the major viral cause of birth defects in the world. In this context, the development of a vaccine to prevent HCMV infection has driven the attention of researchers in the last decades. The most studied candidate to date is a subunit glycoprotein B (gB)-based vaccine. Efficient recombinant protein production platforms are required to produce gB. Recently, plants, and notably Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells, have demonstrated their potential as an alternative platform to mammalian cells for the production of recombinant pharmaceutical proteins. However, several issues are still hampering the development of such plant-based pharmaceuticals. Among them, concerns have arisen regarding the presence of non-human residues on plant N-glycans, that might lead to adverse effects. Moreover, glycoproteins are known to bear a wide variety of N-glycan structures, thus hindering the subsequent quality control, as product homogeneity is often required for the approval of biopharmaceuticals by regulatory authorities. In this work, we produced two HCMV gB variants in two glycoengineered BY-2 cell lines, namely the GnTI-KO and GnTI/FucT-KO lines. N-glycans produced in these cell lines are highly homogeneous, mainly high-mannose and hence practically devoid of non-human residues. Intact gB was found to accumulate in the extracellular medium of the transformed lines in greater proportions when cultured in the D11b medium than in the classical MS medium, thereby suggesting that the D11b medium is more suitable for gB production. Moreover, a first analysis of the N-glycan structures present on the produced gB indicated that the protein mainly bore high-mannose N-glycans, thus matching the expectations. In parallel, a protocol for the thermal shift assay was set up using both commercial immunoglobulins G and gB samples. This method enables the comparison of the thermal stability of proteins in a high-throughput fashion. It was successfully used to study the stability differences between the four immunoglobulin G subclasses. Although it did yield some satisfying results, thermal shift assay performed on gB was found to need further optimization to be fully conclusive.