Precision glycoform engineering: Combining plant and in vitro systems for tailored biopharmaceutical production.

Protein biopharmaceuticals play a key role in providing effective, targeted, and personalized therapies for diverse diseases, while also preventing and mitigating a broad range of infections. N-glycosylation is a key post-translational modification influencing the biological activity of many protein-based therapeutics, yet structure-function relationships of N-glycans remain poorly understood due to challenges in producing homogeneous glycoforms. Current go-to production hosts, mammalian and yeast cells, often yield heterogeneous glycan profiles and require extensive genetic manipulation. Alternative production hosts such as the plant Nicotiana benthamiana, provide more homogeneous glycosylation and flexibility through transient expression, but are limited in the generation of certain glycoforms. In vitro glycoengineering can overcome these limitations but is time consuming and requires expensive resources. In this study, we show that by combining in planta and in vitro glycoengineering strategies, we can quickly produce a wide range of homogeneous glycoforms of pharmaceutical proteins with high mannose, paucimannose, hybrid and complex N-glycan structures. Using N. benthamiana as a transient expression host, we produced two pharmaceutical glycoproteins - the monoclonal antibody rituximab and the helminth vaccine candidate OoASP-1 - and modified them in vitro using Escherichia coli produced glycoenzymes. The combination of these two glycoengineering systems minimizes the amount of time and resources required, while maintaining high glycan homogeneity. This scalable, flexible, and cost-effective platform opens the door to glycan structure-function relationship studies and can support rational design of next-generation biopharmaceuticals.