Erythropoietin N-glycosylation of Therapeutic Formulations Quantified and Characterized: An Interlab Comparability Study of High-Throughput Methods.

Recombinant human erythropoietin (EPO) is a biopharmaceutical frequently used in the treatment of anemia. It is a heavily glycosylated protein with a diverse and complex glycome. EPO N-glycosylation influences important pharmacological parameters, prominently serum half-life. Therefore, EPO N-glycosylation analysis is of the utmost importance in terms of controlling critical quality attributes. In this work, we performed an interlaboratory study of glycoanalytical techniques for profiling and in-depth characterization, namely (1) hydrophilic interaction liquid chromatography with fluorescence detection after 2-aminobenzamide labeling (HILIC-FLD(2AB)) and optional weak anion exchange chromatography (WAX) fractionation and exoglycosidase digestion, (2) HILIC-FLD after procainamide labeling (PROC) optionally coupled to electrospray ionization-MS and (3) matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-MS). All techniques showed good precision and were able to differentiate the unique N-glycosylation profiles of the various EPO preparations. HILIC-FLD showed higher precision, while MALDI-TOF-MS covered the most analytes. However, HILIC-FLD differentiated isomeric N-glycans, i.e., N-acetyllactosamine repeats and O-acetylation regioisomers. For routine profiling, HILIC-FLD methods are more accessible and cover isomerism in major structures, while MALDI-MS covers more minor analytes with an attractively high throughput. For in-depth characterization, MALDI-MS and HILIC-FLD(2AB)/WAX give a similar amount of orthogonal information. HILIC-FLD(PROC)-MS is attractive for covering isomerism of major structures with a significantly less extensive workflow compared to HILIC-FLD(2AB)/WAX.

Serum and Plasma Immunoglobulin G Fc N-Glycosylation Is Stable during Storage.

Immunoglobulin G (IgG) glycosylation is studied in biological samples to develop clinical markers for precision medicine, for example, in autoimmune diseases and oncology. Inappropriate storage of proteins, lipids, or metabolites can lead to degradation or modification of biomolecular features, which can have a strong negative impact on accuracy and precision of clinical omics studies. Regarding the preservation of IgG glycosylation, the range of appropriate storage conditions and time frame is understudied. Therefore, we investigated the effect of storage on IgG Fc N-glycosylation in the commonly analyzed biofluids, serum and plasma. Short-term storage and accelerated storage stability were tested by incubating samples from three healthy donors under stress conditions of up to 50 °C for 2 weeks using -80 °C for 2 weeks as the reference condition. All tested IgG glycosylation features-sialylation, galactosylation, bisection, and fucosylation-remained unchanged up to room temperature as well as during multiple freeze-thaw cycles and exposure to light. Only when subjected to 37 °C or 50 °C for 2 weeks, galactosylation and sialylation subtly changed. Therefore, clinical IgG glycosylation analysis does not rely as heavily on mild serum and plasma storage conditions and timely analysis as many other omics analyses.

Immunoglobulin G Glycoprofiles are Unaffected by Common Bottom-Up Sample Processing.

Immunoglobulin G (IgG) glycosylation is a key post-translational modification in regulating IgG function. It is therefore a prominent target for biomarker discovery and a critical quality attribute of antibody-based biopharmaceuticals. A common approach for IgG glycosylation analysis is the measurement of tryptic glycopeptides. Glycosylation stability during sample processing is a key prerequisite for an accurate and robust analysis yet has hitherto hardly been studied. Especially, acid hydrolysis of sialic acids may be a source for instability. Therefore, we investigated acid denaturation, centrifugal vacuum concentration, and glycopeptide storage regarding changes in the IgG glycosylation profile. Intravenous IgG was analyzed employing imaginable deviations from a reference method and stress conditions. All glycosylation features -sialylation, galactosylation, bisection, and fucosylation-remained unchanged for most conditions. Only with prolonged exposure to acidic conditions at 37 °C, sialylation decreased significantly and subtle changes occurred for galactosylation. Consequently, provided that long or intense heating in acidic solutions is avoided, sample preparation for bottom-up glycoproteomics does not introduce conceivable biases.