Quantitative analysis of trabecular bone tissue cryosections via a fully automated neural network-based approach.

Cryosectioning is known as a common and well-established histological method, due to its easy accessibility, speed, and cost efficiency. However, the creation of bone cryosections is especially difficult. In this study, a cryosectioning protocol for trabecular bone that offers a relatively cheap and undemanding alternative to paraffin or resin embedded sectioning was developed. Sections are stainable with common histological dying methods while maintaining sufficient quality to answer a variety of scientific questions. Furthermore, this study introduces an automated protocol for analysing such sections, enabling users to rapidly access a wide range of different stainings. Therefore, an automated 'QuPath' neural network-based image analysis protocol for histochemical analysis of trabecular bone samples was established, and compared to other automated approaches as well as manual analysis regarding scattering, quality, and reliability. This highly automated protocol can handle enormous amounts of image data with no significant differences in its results when compared with a manual method. Even though this method was applied specifically for bone tissue, it works for a wide variety of different tissues and scientific questions.

Universal ion chromatography method for anions in active pharmaceutical ingredients enabled by computer-assisted separation modeling.

Ion Chromatography (IC) is one of the most widely used methods for analyzing ionic species in pharmaceutical samples. A universal IC method that can separate a wide range of different analytes is highly desired as it can save a lot of time for method development and validation processes. Herein we report the development of a universal method for anions in active pharmaceutical ingredients (APIs) using computer-assisted chromatography modeling tools. We have screened three different IC columns (Dionex IonPac AS28-Fast 4 µm, AS19 4 µm and AS11-HC 4 µm) to determine the best suitable column for universal IC method development. A universal IC method was then developed using an AS11-HC 4 µm column to separate 31 most common anionic substances in 36 mins. This method was optimized using LC Simulator and a model which precisely predicts the retention behavior of 31 anions was established. This model demonstrated an excellent match between predicted and experimental analyte retention time (R2 =0.999). To validate this universal IC method, we have studied the stability of sulfite and sulfide analytes in ambient conditions. The method was then validated for a subset of 29 anions using water and organic solvent/water binary solvents as diluents for commercial APIs. This universal IC method provides an efficient and simple way to separate and analyze common anions in APIs. In addition, the method development process combined with LC simulator modeling can be effectively used as a starting point during method development for other ions beyond those investigated in this study.

Selectivity and Resolving Power of Hydrophobic Interaction Chromatography Targeting the Separation of Monoclonal Antibody Variants.

This study presents a comprehensive investigation of the mechanistic understanding of retention and selectivity in hydrophobic interaction chromatography. It provides valuable insights into crucial method-development parameters involved in achieving chromatographic resolution for profiling molecular variants of trastuzumab. Retention characteristics have been assessed for three column chemistries, i.e., butyl, alkylamide, and long-stranded multialkylamide ligands, while distinguishing column hydrophobicity and surface area. Salt type and specifically chloride ions proved to be the key driver for improving chromatographic selectivity, and this was attributed to the spatial distribution of ions at the protein surface, which is ion-specific. The effect was notably more pronounced on the multialkylamide column, as proteins intercalated between the multiamide polymer strands, enabling steric effects. Column coupling proved to be an effective approach for maximizing resolution between molecular variants present in the trastuzumab reference sample and trastuzumab variants induced by forced oxidation. Liquid chromatography-mass spectrometry (LC-MS)/MS peptide mapping experiments after fraction collection indicate that the presence of chloride in the mobile phase enables the selectivity of site-specific deamidation (N30) situated at the heavy chain. Moreover, site-specific oxidation of peptides (M255, W420, and M431) was observed for peptides situated at the Fc region close to the CH2-CH3 interface, previously reported to activate unfolding of trastuzumab, increasing the accessible surface area and hence resulting in an increase in chromatographic retention.

Hybrid grafted and hyperbranched anion exchangers for ion chromatography.

Novel grafted anion exchangers with covalently bonded hyperbranched functional layers were prepared and evaluated for the separation of monovalent standard inorganic anions and oxyhalides. Preparation of base coating included grafting highly polar N-vinylformamide to the ethylvinylbenzene-divinylbenzene (EVB-DVB) substrate surface in highly polar solvent (methanol) with subsequent hydrolysis of grafted amide polymer in basic media, which resulted in preparation of polymer chains with multiple primary amino groups. Those amino groups were used as attachment points for forming hyperbranched anion-exchange layers using 1,4-butanediol diglycidyl ether and primary mono- or diamine (methylamine or 1,3-diaminopropane, respectively). The effects of hyperbranching reaction cycles number on selectivity were evaluated which revealed that selectivity and capacity can be controlled independently for the covalently bonded stationary phases in contrast to electrostatically bonded phases. It was demonstrated that unlike for electrostatically bonded phases, the intentional increase of crosslink by using primary diamine instead of primary monoamine doesn't cause the shift of selectivity coefficients. It was also shown that crosslink distribution throughout the hyperbranched layer is an important factor determining selectivity of hyperbranched anion exchangers.

A Universal Eluent System for Method Scouting and Separation of Biotherapeutic Proteins by Ion-Exchange, Size-Exclusion, and Hydrophobic Interaction Chromatography.

Characterization and quality control of biotherapeutic proteins commonly require the application of several orthogonal separation techniques in order to establish product identity and purity. Many of the techniques used rely on a buffered aqueous mobile phase system to maintain the native conformation of the protein and its variants. Optimal pH, buffer substance(s), and chromatography methods vary with each protein of interest and result in tedious method development for each new drug product. Linear controlled pH gradient systems from pH 5.6 to pH 10.2 has been shown to provide a global method for the separation of charge variants of monoclonal antibodies. This can be realized using two balanced zwitterionic buffer blends. The pH linearity of the resulting system, with a cation ion exchange column in place, can generate any pH value in this accessible pH range. This study expands the scope of this buffer system and demonstrates its application in conjunction with a quaternary HPLC pump for several analytical techniques: the pH optimization of salt gradient-based anion and cation exchange during method development, as well as performing pH gradient elution. In addition, the same universal buffers are used for hydrophobic interaction and size exclusion chromatography. This eluent system omits the need to prepare different buffers for each method and flushing of the HPLC system between method changes. The implementation of this concept is further demonstrated to allow an automated method scouting approach and selection of different methods that requires minimal manual intervention.

Association of systemic antibody response against polyethylene terephthalate with inflammatory serum cytokine profile following implantation of differently coated vascular prostheses in a rat animal model.

Experimental studies demonstrated antibodies against matrix and coating of polyester-based vascular prostheses. Thus, this study examined associations of these antibodies with serum cytokines (IL-2, IL-4, and IL-10) and local inflammatory reactions. Rats (n = 8/group) intramuscularly received prosthesis segments [PET-C, PET-G, and PET-A groups: polyethylene terephthalate (PET)-based prostheses coated with bovine collagen and gelatin or human serum albumin, respectively; uncoated polytetrafluoroethylene-based (PTFE) prosthesis], with sham-operated controls. Blood was drawn pre-operatively and weekly until day 22. Polymer-specific or coating-specific antibodies and cytokines were detected by enzyme immunoassays, inflammatory reactions were immunohistochemically evaluated on day 23. Polymer-specific antibodies were detected in all PET-groups using uncoated PET as antigenic target, but not for PTFE or controls, coating-specific antibodies only for PET-A. IL-10 was increased in all PET-groups and correlated with polymer-specific antibodies for PET-G and PET-A. IL-2 was increased for PET-A, but overall correlated with PET-specific antibodies. IL-4 remained unchanged in all groups. Intense local inflammatory reactions (ED1+ /ED2+ macrophages and T lymphocytes) were found within all PET-groups, but only minor for PTFE or controls. In conclusion, PET-specific antibodies were associated with increased IL-10 and along with concurrent coating-specific antibodies also with increased IL-2, indicating a specific T cell response. Thus, matrix and/or coating of polymeric vascular prostheses elicit distinct systemic immune reactions, probably influencing local inflammatory reactions.

Local Inflammatory Response after Intramuscularly Implantation of Anti-Adhesive Plasma-Fluorocarbon-Polymer Coated Ti6AI4V Discs in Rats.

Orthopaedic implants and temporary osteosynthesis devices are commonly based on Titanium (Ti). For short-term devices, cell-material contact should be restricted for easy removal after bone healing. This could be achieved with anti-adhesive plasma-fluorocarbon-polymer (PFP) films created by low-temperature plasma processes. Two different PFP thin film deposition techniques, microwave (MW) and radiofrequency (RF) discharge plasma, were applied to receive smooth, hydrophobic surfaces with octafluoropropane (C3F8) or hexafluorohexane (C6F6) as precursors. This study aimed at examining the immunological local tissue reactions after simultaneous intramuscular implantation of four different Ti samples, designated as MW-C3F8, MW-C6F6, RF-C3F8 and Ti-controls, in rats. A differentiated morphometric evaluation of the inflammatory reaction was conducted by immunohistochemical staining of CD68+ macrophages, CD163+ macrophages, MHC class II-positive cells, T lymphocytes, CD25+ regulatory T lymphocytes, NK cells and nestin-positive cells in cryosections of surrounding peri-implant tissue. Tissue samples were obtained on days 7, 14 and 56 for investigating the acute and chronical inflammation (n = 8 rats/group). Implants with a radiofrequency discharge plasma (RF-C3F8) coating exhibited a favorable short- and long-term immune/inflammatory response comparable to Ti-controls. This was also demonstrated by the significant decrease in pro-inflammatory CD68+ macrophages, possibly downregulated by significantly increasing regulatory T lymphocytes.

Effect of High Hydrostatic Pressure on Human Trabecular Bone Regarding Cell Death and Matrix Integrity.

The reconstruction of critical size bone defects is still clinically challenging. Even though the transplantation of autologous bone is used as gold standard, this therapy is accompanied by donor site morbidities as well as tissue limitations. The alternatively used allografts, which are devitalized due to thermal, chemical or physical processing, often lose their matrix integrity and have diminished biomechanical properties. High Hydrostatic Pressure (HHP) may represent a gentle alternative to already existing methods since HHP treated human osteoblasts undergo cell death and HHP treated bone cylinders maintain their mechanical properties. The aim of this study was to determine the biological effects caused by HHP treatment regarding protein/matrix integrity and type of cell death in trabecular bone cylinders. Therefore, different pressure protocols (250 and 300 MPa for 10, 20 and 30 min) and end point analysis such as quantification of DNA-fragmentation, gene expression, SDS-PAGE, FESEM analysis and histological staining were performed. While both protein and matrix integrity was preserved, molecular biological methods showed an apoptotic differentiation of cell death for lower pressures and shorter applications (250 MPa for 10 and 20 min) and necrotic differentiation for higher pressures and longer applications (300 MPa for 30 min). This study serves as a basis for further investigation as it shows that HHP successfully devitalizes trabecular bone cylinders.

Preparation of ion exchange columns with longitudinal stationary phase gradients.

Preparation of columns using electrostatic attachment of anion exchange latex particles with charge density gradients is demonstrated. When such columns are oriented with the highest charge density at the column outlet, the chromatographic performance at low linear velocity is enhanced. When multiple successive charge density gradients are prepared along the length of the column with the highest capacity oriented at the inlet end of the column, significant improvement in chromatographic performance is observed during gradient elution chromatography.

New Insights into the Chromatography Mechanisms of Ion-Exchange Charge Variant Analysis: Dispelling Myths and Providing Guidance for Robust Method Optimization.

Charge variant analysis is a widely used analytical tool in characterization of monoclonal antibodies (mAbs). It depicts the heterogeneity of charge variant forms, some of which may differ by only minor modifications of a single amino acid. The analysis ensures product consistency with no unwanted changes to the protein. With increasing numbers of new mAb drug products emerging in the market, the need for a robust charge variant analysis has intensified. The charge variant profiles often display partially resolved peaks on shoulders of larger peaks. This puts considerably more pressure on the robustness of the method to maintain the suboptimum selectivity. New products and techniques have emerged to address these requirements, in addition to the pre-existing older methods that may not have been optimized correctly in the past. This has led to some confusion as to the best approach and strategies in optimization of charge variant analysis. We show studies from several different approaches using on-line pH monitoring to check the performance characteristics of the methods. This has led to new insights on the interactions between the protein, column, and buffer constituents. We dispel some inaccurate assumptions about the different ion-exchange elution mechanisms and suggest ways to develop high-throughput methods that remain robust and of high resolution. Streamlined automatable method development tools are presented that will result in more efficient method optimization. The mechanisms behind poor chromatography design have provided an alternative explanation behind some methods failing when in the QC laboratories.

Localised quantitative structure-retention relationship modelling for rapid method development in reversed-phase high performance liquid chromatography.

Quantitative structure-retention relationships (QSRR) predicting the values of solute "hydrophobicity" coefficient η' in the approximate hydrophobic subtraction model (HSM) can be used to predict retention times of compounds on numerous reversed-phase (RP) columns, provided that column parameters on the corresponding stationary phases are available. In the present study, we propose a new dual clustering-based localised QSRR approach, combining P-ratio clustering (where P is the octanol-water partition coefficient) with second dominant interaction (SDI)-based clustering, to produce predictive models with an acceptable level of prediction accuracy for in silico column scoping in RP method development. QSRR models for η' values were derived for 49 compounds out of 63 in a dataset extracted from the literature, where retention data were measured under one isocratic mobile phase condition (i.e., acetonitrile-water, 50:50 [v/v]). These models gave a predictive squared correlation coefficient Qext(F2)2 of 0.83 and a root mean square error of prediction (RMSEP) of 0.14. For the modelling, a genetic algorithm-partial least square regression (GA-PLS) approach was performed using the η' values and their relevant molecular descriptors. The corresponding retention times were predicted by applying the predicted η' values of the models and the stationary phase "hydrophobicity" parameter H values for the corresponding columns to the approximate HSM, resulting in excellent accuracy and predictability (Qext(F2)2 of 0.90 and RMSEP of 0.72 min). The established QSRR approach was experimentally verified for six Thermo Scientific columns (Acclaim™ 120 C18, Acclaim Polar Advantage, Acclaim Polar Advantage II, Accucore™ aQ, Accucore Phenyl-X, and Hypersil Gold C18 columns) using two types of datasets. The first dataset consisted of eight model compounds extracted from the original dataset and retention time predictions for those compounds were then evaluated on the above columns. The result showed good agreement between predicted and observed retention times with an acceptable error in retention time predictions (slope of 0.97, Qext(F2)2 of 0.95, a mean absolute error (MAE) of 0.43 min and RMSEP of 0.61 min). The second dataset included eight test compounds not included in the original dataset, which were all classified into the η' cluster by applying a Tanimoto similarity (TS) threshold of 0.7. Similarly, predicted retention times of the test compounds were compared with their corresponding observed retention times, resulting in acceptable retention time predictions with the slope of 0.99, Qext(F2)2 of 0.93 and RMSEP of 0.52 min. Comparisons of resolution values between columns were utilised to select the most suitable columns for separations of the compounds in the respective test sets. Actual chromatograms obtained on the chosen columns showed the feasibility for effective column scoping without experimentation on numerous RP stationary phases available in the USP website, based on the predicted resolution values.

Ultra-High-Pressure Ion Chromatography with Suppressed Conductivity Detection at 70 MPa Using Columns Packed with 2.5 µm Anion-Exchange Particles.

The use of ultrahigh pressures in combination with columns packed with 2.5 µm microporous and supermacroporous (perfusive) stationary phase particles coated with nanobeads has been successfully explored in ion chromatography with online eluent generation and suppressed conductivity detection. Isocratic separations of inorganic anions and organic acids yielding reduced plate heights as low as 2.1 were achieved, corresponding to efficiencies up to 190000 plates/m, using an optimized system configuration with respect to injection parameters, considering volume and mass loadability, and extra-column dispersion. Viscous-heating effects have been assessed for PEEK-lined stainless steel columns operated at 70 MPa, and effects of thermal gradients on separation efficiency and retention are demonstrated. Whereas the PEEK-lined column hardware acts to some extent as an insulator, a 10% increase in plate number could be obtained when applying a still-air column oven configuration. In the forced-air mode, an increase in retention was observed for polyvalent ions. Finally, the kinetic performance limits of ultrahigh-pressure ion chromatography applying 2.5 µm particle-packed columns operated at 70 MPa were compared to conventional ion-chromatography technology using columns packed with 4 µm particles operated at a maximum pressure of 35 MPa. Downscaling the particle size and increasing the operating pressure led to a maximum time gain with a factor of 3.4, without compromising separation efficiency (N = 10000).

Retention prediction using quantitative structure-retention relationships combined with the hydrophobic subtraction model in reversed-phase liquid chromatography.

The hydrophobic subtraction model (HSM) combined with quantitative structure-retention relationships (QSRR) methodology was utilized to predict retention times in reversed-phase liquid chromatography (RPLC). A selection of new analytes and new RPLC columns that had never been used in the QSRR modeling process were used to verify the proposed approach. This work is designed to facilitate early prediction of co-elution of analytes in pharmaceutical drug discovery applications where it is advantageous to predict whether impurities might be co-eluted with the active drug component. The QSRR models were constructed through partial least squares regression combined with a genetic algorithm (GA-PLS) which was employed as a feature selection method to choose the most informative molecular descriptors calculated using VolSurf+ software. The analyte hydrophobicity coefficient of the HSM was predicted for subsequent calculation of retention. Clustering approaches based on the local compound type and the local second dominant interaction were investigated to select the most appropriate training set of analytes from a larger database. Predicted retention times of five new compounds on five new RPLC C18 columns were compared with their measured retention times with percentage root-mean-square errors of 15.4 and 24.7 for the local compound type and local second dominant interaction clustering methods, respectively.

Dual Electrolytic Eluent Generation for Oligosaccharides Analysis Using High-Performance Anion-Exchange Chromatography.

The research on oligosaccharides is growing and gaining in importance at a rapid pace. The efforts to understand their bioactivity and to develop new products based on oligosaccharides in biotherapeutics and food industry require effective and reliable tools for analysis of oligosaccharides. Here we present a dual electrolytic eluent generation platform for the analysis of oligosaccharides by high-performance anion-exchange liquid chromatography (HPAE) in both analytical and capillary column formats. The system consists of one eluent generator producing methanesulfonic acid (MSA) connected in series with a second eluent generator producing potassium hydroxide (KOH). Through manipulating the concentration output of both eluent generators, chromatographic performance comparable to that obtained using the conventional sodium acetate/sodium hydroxide (NaOAc/NaOH) eluents is achieved using the electrolytically generated potassium methanesulfonate/potassium hydroxide (KMSA/KOH) eluent. This platform utilizes deionized water as the only carrier stream through a single isocratic pump, overcomes the various drawbacks associated with manually prepared NaOAc/NaOH eluents, and offers an easy to use, simplified operation solution for oligosaccharides profiling with increased precision and accuracy.

A new suppressor design for low noise performance with carbonate eluents for Ion Chromatography.

Carbonate and bicarbonate based eluents have been applied for ion analysis from the inception of ion chromatography. The product of suppression with carbonate and/or bicarbonate eluent is carbonic acid which is weakly dissociated and tends to outgas. While the act of suppression enhanced the signal for fully dissociated ions and lowered the background to a weakly dissociated level, the overall noise performance, however, varied depending on the suppression mechanism. Chemical suppression with a membrane suppressor yielded low noise performance with carbonate and/or bicarbonate eluents. Electrolytic suppression, on the other hand, resulted in a relatively higher noise with carbonate based eluents when compared to chemical suppression. In this work, we investigated the root cause of noise with electrolytic suppressors and carbonate based eluents. Further, a new electrolytic suppressor design based on a three-electrode design is discussed in this paper and provided low noise performance with carbonate and/or bicarbonate eluents.

Retention Index Prediction Using Quantitative Structure-Retention Relationships for Improving Structure Identification in Nontargeted Metabolomics.

Structure identification in nontargeted metabolomics based on liquid-chromatography coupled to mass spectrometry (LC-MS) remains a significant challenge. Quantitative structure-retention relationship (QSRR) modeling is a technique capable of accelerating the structure identification of metabolites by predicting their retention, allowing false positives to be eliminated during the interpretation of metabolomics data. In this work, 191 compounds were grouped according to molecular weight and a QSRR study was carried out on the 34 resulting groups to eliminate false positives. Partial least squares (PLS) regression combined with a Genetic algorithm (GA) was applied to construct the linear QSRR models based on a variety of VolSurf+ molecular descriptors. A novel dual-filtering approach, which combines Tanimoto similarity (TS) searching as the primary filter and retention index (RI) similarity clustering as the secondary filter, was utilized to select compounds in training sets to derive the QSRR models yielding R2 of 0.8512 and an average root mean square error in prediction (RMSEP) of 8.45%. With a retention index filter expressed as ±2 standard deviations (SD) of the error, representative compounds were predicted with >91% accuracy, and for 53% of the groups (18/34), at least one false positive compound could be eliminated. The proposed strategy can thus narrow down the number of false positives to be assessed in nontargeted metabolomics.

Ion exchange column capacities. Predicting retention behavior of open tubular columns coated with the same phase.

We discuss the reported capacities of available packed ion exchange columns and the different methods used for their measurement. We outline basic considerations related to both packed and open tubular columns based on ion exchange latex particles. There is a large body of information covering the retention behavior of packed ion exchange columns based on ion exchange latex particles. We propose a parameter γiex, which is the ion exchange capacity of a column (packed or open tubular) per unit liquid volume present in the column (including accessible volume within pores) and show that the retention factor for any given ion is directly related to γiex. On this basis, if based on the same type of latex, the behavior of one type of column can be reasonably predicted from the known behavior of the other, even when the absolute capacities differ by more than 5 orders of magnitude.

Electrostatically attached highly branched anion-exchange phases derived from diamines and diepoxides.

A new synthesis approach for the preparation of highly branched anion exchange materials utilizing diamine and diepoxide reagents is described. Unlike previously reported condensation polymers prepared from primary amine and diepoxide reagents, anion exchange polymers prepared from diamines and diepoxide reagents exhibit exceptionally low affinity for polyvalent ions. Use of anion-exchange materials synthesized utilizing this new synthetic method for the analysis of common inorganic anions is demonstrated.

Retention prediction in reversed phase high performance liquid chromatography using quantitative structure-retention relationships applied to the Hydrophobic Subtraction Model.

Quantitative Structure-Retention Relationships (QSRR) methodology combined with the Hydrophobic Subtraction Model (HSM) have been utilized to accurately predict retention times for a selection of analytes on several different reversed phase liquid chromatography (RPLC) columns. This approach is designed to facilitate early prediction of co-elution of analytes, for example in pharmaceutical drug discovery applications where it is advantageous to predict whether impurities might be co-eluted with the active drug component. The QSRR model utilized VolSurf+ descriptors and a Partial Least Squares regression combined with a Genetic Algorithm (GA-PLS) to predict the solute coefficients in the HSM. It was found that only the hydrophobicity (η'H) term in the HSM was required to give the accuracy necessary to predict potential co-elution of analytes. Global QSRR models derived from all 148 compounds in the dataset were compared to QSRR models derived using a range of local modelling techniques based on clustering of compounds in the dataset by the structural similarity of compounds (as represented by the Tanimoto similarity index), physico-chemical similarity of compounds (represented by log D), the neutral, acidic, or basic nature of the compound, and the second dominant interaction between analyte and stationary phase after hydrophobicity. The global model showed reasonable prediction accuracy for retention time with errors of 30 s and less for up to 50% of modeled compounds. The local models for Tanimoto, nature of the compound and second dominant interaction approaches all exhibited prediction errors less than 30 s in retention time for nearly 70% of compounds for which models could be derived. Predicted retention times of five representative compounds on nine reversed-phase columns were compared with known experimental retention data for these columns and this comparison showed that the accuracy of the proposed modelling approach is sufficient to reliably predict the retention times of analytes based only on their chemical structures.

High Performance Anion Exchange and Hydrophilic Interaction Liquid Chromatography Approaches for Comprehensive Mass Spectrometry-Based Characterization of the N-Glycome of a Recombinant Human Erythropoietin.

Comprehensive characterization of the N-glycome of a therapeutic is challenging because glycans may harbor numerous modifications (e.g., phosphorylation, sulfation, sialic acids with possible O-acetylation). The current report presents a comparison of two chromatographic platforms for the comprehensive characterization of a recombinant human erythropoietin (rhEPO) N-glycome. The two platforms include a common workflow based on 2-AB-derivatization and hydrophilic interaction chromatography (HILIC) and a native N-linked glycan workflow employing high performance anion exchange (HPAE) chromatography. Both platforms were coupled to an Orbitrap mass spectrometer, and data dependent HCD fragmentation allowed confident structural elucidation of the glycans. Each platform identified glycans not revealed by the other, and both exhibited strengths and weaknesses. The reductive amination based HILIC workflow provided better throughput and sensitivity, had good isomer resolution, and revealed the presence of O-acetylated sialic acids. However, it exhibited poor performance toward phosphorylated glycans and did not reveal the presence of sulfated glycans. Furthermore, reductive amination introduced dehydration artifacts and modified the glycosylation profile in the rhEPO glycome. Conversely, HPAE provided unbiased charge classification (sialylation levels), improved isomer resolution, and revealed multiple phosphorylated and sulfated structures, but delivered lower throughput, had artifact peaks due to epimer formation, and loss of sialic acid O-acetylation. The MS2 based identification of phosphorylated and sulfated glycans was not possible in HILIC mode due to their poor solubility caused by the high acetonitrile concentrations employed at the beginning of the gradient. After analyzing the glycome by both approaches and determining the glycans present, a glycan library was created for site specific glycopeptide analyses. Glycopeptide analyses confirmed all the compositions annotated by the combined use of 2-AB- and native glycan workflows and provided site specific location of the glycans. These two platforms were complementary and in combination delivered a more thorough and comprehensive characterization of the rhEPO N-glycome, supporting regulatory conformance for the pharmaceutical industry.