Precision Glycoform engineering: Combining Plant and in vitro Systems for Biopharmaceutical production

The global market of biopharmaceuticals has seen substantial growth over the years and has reached an estimated $300 billion revenue in 2023. Approximately 70% of these biopharmaceuticals are glycosylated, underscoring the critical role of protein glycosylation. In addition, a plethora of studies indicate that the glycoform attached to biopharmaceuticals is crucial for their functionality, making it necessary to be able to synthesize specific and homogeneous glycoforms in vivo or in vitro. Nicotiana benthamiana is a versatile biopharmaceutical production system that can generate a range of homogeneously glycosylated proteins. By transiently expressing non-native Golgi localized glyco-enzymes, N-glycans can be tailored in vivo into the desired glycoform without adverse growth effects. Although the plant system is an excellent system for biopharmaceutical production, it also has its drawbacks. Plant native glycoenzymes prohibit the generation of specific glycoforms. To circumvent this, we set up an in vitro glycosylation system with Escherichia coli produced glycoenzymes, lacking their transmembrane region. By combining both systems, we can produce a range of homogeneous glycoforms, ranging from high mannose, paucimannose, hybrid and complex glycan structures. This work paves the way for establishing a platform to produce homogeneously glycosylated biopharmaceuticals with maximum efficacy, while also addressing a variety of biological questions.