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Early diagnosis and dietary treatment do not prevent long-term complications, which mostly affect the central nervous system in classical galactosemia patients. The clinical outcome of patients is highly variable, and there is an urgent need for prognostic biomarkers. The aim of this study was first to increase knowledge on the natural history of classical galactosemia by studying a cohort of patients with varying geno- and phenotypes and second to study the association between clinical outcomes and two possible prognostic biomarkers. In addition, the association between abnormalities on brain MRI and clinical outcomes was investigated. Classical galactosemia patients visiting the galactosemia expertise outpatient clinic of the Amsterdam University Medical Centre were evaluated according to the International Classical Galactosemia guideline with the addition of an examination by a neurologist, serum immunoglobulin G N -glycan profiling and a brain MRI. The biomarkers of interest were galactose-1-phosphate levels and N -glycan profiles, and the clinical outcomes studied were intellectual outcome and the presence or absence of movement disorders and/or primary ovarian insufficiency. Data of 56 classical galactosemia patients are reported. The intellectual outcome ranged from 45 to 103 (mean 77 ± 14) and was <85 in 62%. Movement disorders were found in 17 (47%) of the 36 tested patients. In females aged 12 years and older, primary ovarian insufficiency was diagnosed in 12 (71%) of the 17 patients. Significant differences in N -glycan peaks were found between controls and patients. However, no significant differences in either N -glycans or galactose-1-phosphate levels were found between patients with a poor (intellectual outcome < 85) and normal intellectual outcome (intellectual outcome ≥ 85), and with or without movement disorders or primary ovarian insufficiency. The variant patients detected by newborn screening, with previously unknown geno- and phenotypes and currently no long-term complications, demonstrated significantly lower galactose-1-phospate levels than classical patients ( P  < 0.0005). Qualitative analysis of the MRI's demonstrated brain abnormalities in 18 of the 21 patients, more severely in patients with a lower intellectual outcome and/or with movement disorders. This study demonstrates a large variability in clinical outcome, which varies from a below average intelligence, movement disorders and in females primary ovarian insufficiency to a normal clinical outcome. In our cohort of classical galactosemia patients, galactose-1-phosphate levels and N -glycan variations were not associated with clinical outcomes, but galactose-1-phosphate levels did differentiate between classical and variant patients detected by newborn screening. The correlation between brain abnormalities and clinical outcome should be further investigated by quantitative analysis of the MR images. The variability in clinical outcome necessitates individual and standardized evaluation of all classical galactosemia patients., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Glycosylation of the Fc region of IgG has a profound impact on the safety and clinical efficacy of therapeutic antibodies. While the biantennary complex-type oligosaccharide attached to Asn297 of the Fc is essential for antibody effector functions, fucose and outer-arm sugars attached to the core heptasaccharide that generate structural heterogeneity (glycoforms) exhibit unique biological activities. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for the development and quality control of therapeutic antibodies, and glycan profiles of the Fc are recognized as critical quality attributes. In the past decade our understanding of the influence of glycosylation on the structure/function of IgG-Fc has grown rapidly through X-ray crystallographic and nuclear magnetic resonance studies, which provides possibilities for the design of novel antibody therapeutics. Furthermore, the chemoenzymatic glycoengineering approach using endoglycosidase-based glycosynthases may facilitate the development of homogeneous IgG glycoforms with desirable functionality as next-generation therapeutic antibodies. Thus, the Fc glycans are fertile ground for the improvement of the safety, functionality, and efficacy of therapeutic IgG antibodies in the era of precision medicine.

Complex carbohydrates are rapidly becoming excellent biomarker candidates because of their high sensitivity to pathological changes. However, the discovery of clinical glycobiomarkers has been slow, due to the scarcity of high-throughput glycoanalytical workflows that allow rapid glycoprofiling of large clinical sample sets. To generate high-quality quantitative glycomics data in a high-throughput fashion, we have developed a robotized platform for rapid serum-based N-glycan sample preparation. The sample preparation workflow features a fully automated, rapid glycoprotein denaturation followed by sequential enzymatic glycan release, glycan purification on solid-supported hydrazide and fluorescent labelling. This allows accurate glycan quantitation by ultra-high performance liquid chromatography (UPLC). The sample preparation workflow was automated using an eight-channel Hamilton Robotics liquid handling workstation, allowing the preparation of almost 100 samples in 14 hours with excellent reproducibility and thus should greatly facilitate serum-based glyco-biomarker discovery.