Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System.

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the "bead array in a single tip (BIST)" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving "glyco-qualified" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of <10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.