Equivalence assessment of biotherapeutics with N- and O-glycosylation sites by sequential intact glycoform mass spectrometry (IGMS).

Glycosylation is a crucial attribute for biotherapeutics with significant impacts on quality, stability, safety, immunogenicity, pharmacokinetics, and efficacy. Therefore, to ensure consistent glycosylation, a systematic review of biotherapeutics is absolutely required including the variable glycan structure (micro-heterogeneity) and different occupancy at individual site (macro-heterogeneity) from drug design to upstream and downstream bioprocesses. Various methods have been used for glyco-characterization of biotherapeutics at the glycan, glycopeptide, and intact protein levels. In particular, intact protein analysis is considered a facile and rapid glycoform monitoring approach used throughout the product development lifecycle to determine suitable glycosylation lead candidates and reproducible product quality. However, intact glycoform characterization of diverse and complex biotherapeutics with multiple N- and O-glycosylation sites can be very challenging. To address this, a robust analytical platform that enables rapid and accurate characterization of a biotherapeutics with highly complex multiple glycosylation using two-step intact glycoform mass spectrometry has been developed. We used darbepoetin alfa, a second-generation EPO bearing multiple N- and O-glycosylation sites, as a model biotherapeutics to obtain integrated information on glycan heterogeneity and site occupancy through step-by-step MS of intact protein and enzyme-treated protein. In addition, we performed a comparative assessment of the heterogeneity from different products, confirming that our new method can efficiently evaluate glycosylation equivalence. This new strategy provides rapid and accurate information on the degree of glycosylation of a therapeutic glycoprotein with multiple glycosylation, which can be used to assess glycosylation similarity between batches and between biosimilar and reference during development and production.

Advanced assessment through intact glycopeptide analysis of Infliximab's biologics and biosimilar.

Characterization of therapeutic monoclonal antibodies (mAbs) represents a major challenge for analytical sciences due to their heterogeneity associated with post-translational modifications (PTMs). The protein glycosylation requires comprehensive identification, which could influence on the mAbs' structure and their function. Here, we demonstrated high-resolution tandem mass spectrometry with an ultra-high-performance liquid chromatography for characterization and comparison between biologics and biosimilar of infliximab at an advanced level. Comparing the N- and O-glycopeptides profiles, a total of 49 and 54 glycopeptides was identified for each product of the biologics and biosimilar, respectively. We also discovered one novel N-glycosylation site at the light chain from both biopharmaceuticals and one novel O-glycopeptide at the heavy chain from only biosimilar. Site-specific glycopeptide analysis process will be a robust and useful technique for evaluating therapeutic mAbs and complex glycoprotein products.

Comparative proteomics: assessment of biological variability and dataset comparability.

BACKGROUND: Comparative proteomics in bacteria are often hampered by the differential nature of dataset quality and/or inherent biological deviations. Although common practice compensates by reproducing and normalizing datasets from a single sample, the degree of certainty is limited in comparison of multiple dataset. To surmount these limitations, we introduce a two-step assessment criterion using: (1) the relative number of total spectra (R TS ) to determine if two LC-MS/MS datasets are comparable and (2) nine glycolytic enzymes as internal standards for a more accurate calculation of relative amount of proteins. Lactococcus lactis HR279 and JHK24 strains expressing high or low levels (respectively) of green fluorescent protein (GFP) were used for the model system. GFP abundance was determined by spectral counting and direct fluorescence measurements. Statistical analysis determined relative GFP quantity obtained from our approach matched values obtained from fluorescence measurements. RESULTS: L. lactis HR279 and JHK24 demonstrates two datasets with an R TS value less than 1.4 accurately reflects relative differences in GFP levels between high and low expression strains. Without prior consideration of R TS and the use of internal standards, the relative increase in GFP calculated by spectral counting method was 3.92 ± 1.14 fold, which is not correlated with the value determined by the direct fluorescence measurement (2.86 ± 0.42 fold) with the p = 0.024. In contrast, 2.88 ± 0.92 fold was obtained by our approach showing a statistically insignificant difference (p = 0.95). CONCLUSIONS: Our two-step assessment demonstrates a useful approach to: (1) validate the comparability of two mass spectrometric datasets and (2) accurately calculate the relative amount of proteins between proteomic datasets.