MSc Chemistry Analytical Sciences

Master Thesis

Capillary electrophoresis; one technique to completely characterize biopharmaceuticals

by

Fleur Kuijpers 11034556

March 2018 12 credits

January 2018 – March 2018

Supervisor/Examiner: Examiner: Prof. dr. G. W. Somsen Dr. H. Lingeman

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Abstract Biopharmaceuticals – therapeutically active biomolecules – have become an important class of drugs. Over 260 variants are on the market nowadays. They have proven to be efficient in the treatment against many diseases, such as cancer, diabetes, autoimmune disorders, and anemia. Moreover, they show an increased efficacy, specificity, and less side effects compared to low-molecular weight therapeutics. As a result of expression, purification, and storage processes, biopharmaceuticals can undergo post-translational modifications, degradation, and aggregation, which can highly affect the efficacy and stability of the product. Therefore, the product must be subjected to characterization analyses in order to monitor the physical, chemical, biological, or microbiological properties of the product. Additionally, biosimilars – which are developed in order to function equally compared to the structure and clinical properties of the original product – need to be extensively checked before they can enter the market. This review describes the ability of capillary electrophoresis (CE) to completely characterize a biopharmaceutical in terms of their structural and physicochemical properties, based on literature from the last two years. We describe the following characterization analysis categories ordered from intact to amino acid level: size variants, charge variants, aggregates, N-linked glycosylation, O-linked glycosylation, and amino acid sequence. For the analysis of charge and size-based variants, CE is extensively developed allowing even more successful separations compared to chromatographic techniques. The same applies for analyzing N-glycans and O-glycans. The following CE approaches have been performed regarding these characterization analyses: capillary isoelectric focusing (CIEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), ultra-high voltage capillary electrophoresis (UHVCE), open-tubular capillary electrochromatography (OT-CEC), CIEF-CZE, and CZE-CZE. Moreover, CZE and CGE were also accomplished in microfluidic setup. The CE-based separation techniques have been coupled to either UV/-vis, laser-induced fluorescence, or MS detectors. The main reason for the wide application of CE in this field is its resolving power based on charge and/or size, which has proven to be essential for post-translational modifications that often affect the electrophoretic mobility of a compound. Determination of aggregates and amino acid sequencing is mainly performed using other techniques than CE, although CE could also be applied for these analyses. Thus, CE can be considered as a suitable technique to completely characterize biopharmaceuticals.

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List of abbreviations ACN Acetonitrile ADC Antibody drug conjugate AF4 Asymmetric flow field flow fractionation AUC Analytical ultracentrifugation BFS Bare-fused silica BGE Background electrolyte CD Circular dichroism CE Capillary electrophoresis CEC Capillary electrochromatography CEX Cation exchange chromatography CGE Capillary gel electrophoresis CID Collision-induced dissociation CIEF Capillary isoelectric focusing CMC Critical micellar concentration CQA Critical quality attribute CV Coefficient of variation CZE Capillary zone electrophoresis DAR Drug-to-antibody ratio DDT Dithiothreitol DS Dextran sulfate DSC Differential scanning calorimetry EMA European Medicines Agency EOF Electroosmotic flow EPO Erythropoietin ESI Electrospray-ionization FA Formic acid FDA Food and Drug Administration FT-IR Fourier transform infrared HAc Acetic acid HCD Higher-energy collisional dissociation HIC Hydrophobic interaction chromatography HILIC Hydrophilic interaction liquid chromatography HILIC-FD Hydrophilic interaction liquid chromatography fluorescence detection HMWI High molecular weight isoform HOS Higher order structure HPC Hydroxypropyl cellulose HPLC High performance liquid chromatography HPMC Hydroxypropylmethyl cellulose HP-SEC High performance size exclusion chromatography IEF Isoelectric focusing IEX Ion exchange chromatography IgG Immunoglobulin LIF Laser-induced fluorescence mAb Monoclonal antibody MALDI Matrix-assisted laser desorption/ionization MCGE Microchip capillary gel electrophoresis MEKC Micellar electrokinetic capillary chromatography

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MES 2-(N-morpholino)ethanesulfonic acid MS Mass spectrometry MWCO Molecular weight cut-off m/z Mass/charge ratio NMR Nuclear magnetic resonance OT-CEC Open tubular capillary electrochromatographic PB Polybrene PCA Principal component analysis PEG Polyethylene glycol PGC Porous graphitized carbon PTM Post-translational modification QqTOF Quadrupole-quadrupole time of flight QTOF Quadrupole time-of-flight rFIX Recombinant coagulation factor IX rhEPO Recombinant human erythropoietin RP Reversed phase RSD Relative standard deviation SDS-PAGE Dodecyl sulfate polyacrylamide gel electrophoresis SEC Size exclusion chromatography SHS Sodium hexadecyl sulfate SPE Solid phase extraction TETA Triethylene tetramine TFA Trifluoro acetic acid UHV Ultra-high voltage UHVCE Ultra-high voltage capillary electrophoresis UPLC Ultra performance liquid chromatography UV Ultraviolet

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Table of Contents Abstract ................................................................................................................................3

1. Introduction .................................................................................................................7

2. Technical aspects........................................................................................................10 2.1 Capillary Electrophoresis (CE) in relation to analysis of biopharmaceuticals .................. 10 2.2 Optical detectors.................................................................................................................. 12 2.3 Mass spectrometric detection .............................................................................................. 12

3. Intact biopharmaceutical analysis .............................................................................14 3.1 Size variants and fragments of the biopharmaceutical ...................................................... 14 3.2 Charge variants ................................................................................................................... 15 3.3 Aggregates ........................................................................................................................... 23

4. Peptide and PTM analysis .........................................................................................25 4.1 N-linked glycan composition ............................................................................................... 25 4.2 Glycosylation site ID and O-linked analysis ....................................................................... 29

5. Amino acid sequencing ..............................................................................................32

6. Discussion ...................................................................................................................34

7. Conclusion ..................................................................................................................36

References ..........................................................................................................................37

Appendix I: Overview methods applied regarding characterization analysis during the last two years......................................................................................................................42

Intact biopharmaceutical analysis ............................................................................................ 42 Peptide and PTM analyses ........................................................................................................ 44 Amino acid sequencing ............................................................................................................. 45

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1. Introduction Biopharmaceuticals are considered therapeutically active biomolecules produced by native biological tissues or using living cells (1). They come in a broad range of variants of which protein-based drugs – such as vaccines, blood factors, cytokines, monoclonal antibodies (mAbs), hormones, and fusion proteins – are the most common. Since the production of the first biopharmaceutical insulin for the treatment of diabetes in 1982, protein therapeutics have gained enormous interest resulting in over 260 unique biopharmaceuticals on the market nowadays (1). These products can be highly efficient in the treatment against various diseases, such as cancer, diabetes, autoimmune disorders, and anemia (3, 4). Additionally, they have proven to be very beneficial due to their increased efficacy and specificity compared to low-molecular weight drugs and they show less adverse reactions due to their structural similarity to endogenous proteins (4). During the expression, purification, and long-term storage of biopharmaceuticals, the product can undergo post-translational modifications (PTMs), degradation, and aggregation, which can largely affect the properties of these products (4). As a result of all possible PTMs that can occur – such as glycosylation, oxidation, phosphorylation, disulfide bond formation, and deamidation (5) – multiple variants of the original biopharmaceutical can be created containing small heterogeneities related to charge, size, and structure (4, 7, 8). Moreover, the type and abundance of the modifications are influenced by the production cell type, culture conditions, purification strategy, formulation, and storage device format (1). Besides PTMs, the biopharmaceutical can also be subjected to aggregation during reconstitution (3). Considering all the changes that can occur to the therapeutic compound, there is constant need to determine and monitor structural and stability changes during the development of biopharmaceuticals in order to maintain the quality of the product. As a subclass of biopharmaceuticals, biosimilars are produced in order to function similarly compared to the structure and clinical properties of the original product, in terms of quality, safety, and efficacy (9, 6, 7). Once a patent is obtained for a certain biopharmaceutical, the manufacturing process of biosimilars becomes increasingly interesting. As a result, the need for analytical techniques allowing characterization of biosimilars will also rise, since their properties must be compared to the properties of the reference product. In the context of comparison analyses there are two types of performances: external and internal comparison studies. Before the biosimilar can be introduced to the market, the product must be subjected to extensive comparability studies in order to prove the high similarities with the reference product according to the European Medicines Agency (EMA) or the US Food and Drug Administration (FDA). This analysis is considered the external comparison study. In contrast, the internal study consists of the analysis regarding the pre- and post-manufacturing process properties of the biosimilar, since many modifications of the product can occur during the production process(10). Both external and internal comparability studies thus are the basis for a potential approval. To determine the quality of the biosimilar and the originator, its critical quality attributes (CQAs) need to be investigated over an extended period (8). CQAs are described as the physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality (11). Monitoring variations of the CQAs during the manufacturing process is essential to preserve the quality of the biosimilar. The quality of a biosimilar – or the characterization of the protein therapeutic – can be monitored on different levels (Figure 1). Starting with the intact biopharmaceutical, the protein’s secondary, tertiary, and quaternary structure reveals the three-dimensional shape of

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the molecule, which is decisive for the molecule’s biological function (12). In solution, they tend to self-associate or form complexes with other structures present in the sample partly dependent on environmental conditions. Analyses regarding the intact biopharmaceutical, include the research to aggregates and other complexes within the sample, protein fold, and protein conformation. Another category of analyses yield information about the peptide level of the protein therapeutic. This includes analysis of PTMs and peptide sequencing for example. Finally, amino acid sequencing reveals information about the primary structure of the protein.

Figure 1: The different protein levels regarding the analysis of biopharmaceuticals. Intact biopharmaceutical, protein, peptide and PTMs (post-translational modifications), and AA (amino acid) level.

To overcome the challenges of characterizing biopharmaceuticals on all levels, current research efforts are attempting to develop new analytical technologies which contribute to a broader understanding of the product’s quality and impact in terms of safety and efficacy. In that context, the quality of the biopharmaceutical is defined as its suitability for the intended clinical performance. Many analytical techniques have been proposed to improve characterization analysis, including multiple strategies. For example, reversed-phase liquid chromatography is widely used due to its robustness, reproducibility, and wide applicability regarding hydrophobic interaction (13). High performance liquid chromatography (HPLC) systems can be conveniently coupled to mass spectrometry (MS) devices with an electrospray-ionization (ESI) interface allowing mass identification. Besides HPLC approaches, more techniques have been introduced – such as capillary electrophoresis (CE), analytical ultracentrifugation (AUC), asymmetric flow field flow fractionation (AF4), differential scanning calorimetry (DSC), X-ray crystallography, nuclear magnetic resonance (NMR), circular dichroism (CD), fluorescence and Fourier transform infrared (FT-IR) spectroscopy (3) – allowing complete characterization of biopharmaceuticals when complimentary performed. CE has become increasingly applicable in the biopharmaceutical industry because of its separation efficiency, short analysis time, selectivity, and the requirement of low sample

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volumes. Since CE is based on charge differences between compounds, the changing electrophoretic mobility of biopharmaceuticals due to PTMs makes CE a very valuable technique in this field (14). Moreover, CE yields structure-related information about the analyzed therapeutic, due to the charged-based separation mechanism (9). Another factor that makes CE even more useful, is the ability to connect it with MS allowing isoform identification and PTM elucidation. In this review, we discuss the ability of CE approaches to completely characterize biopharmaceuticals in terms of their structural and physicochemical properties. The chapters include the following characterization analysis categories ordered from intact to amino acid level: aggregates, size variants, charge variants, N-linked glycosylation, O-linked glycosylation, and amino acid sequence. We will mainly focus on the performance of the electrophoretic strategy compared to non-electrophoretic strategies, disregarding the accomplished purification, derivatization and sample preparation procedures. As a result of the fast developments in this field (15), this review covers the literature over the last two years.

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2. Technical aspects 2.1 Capillary Electrophoresis (CE) in relation to analysis of biopharmaceuticals From the first moment that CE was demonstrated by Hjerten in 1967, it has grown to an extensively applied separation technique (16). Using an electric field over a solution, they allowed separation of charged compounds. This publication has proven to be a fundament in analytics, since CE is still widely used in the field of biopharma. After the introduction of CE, several approaches within CE have been developed. In 1984, Terabe proposed a novel technique enabling separation of neutral compounds using micellar electrokinetic capillary chromatography (MEKC) (17). This invention was quickly followed up by the development of capillary isoelectric focusing (CIEF) by Hjerten in 1985 (18). CIEF is especially applied for the separation of proteins. In the late 1980’s another approach had been introduced by the group of Karger, who used capillary gel electrophoresis (CGE) for the separation of DNA fragments and proteins (19). A couple of years later, in 1994, Smith and Evans introduced the first practical application of capillary electrochromatography (CEC) (16). CE includes several electrophoretic separation strategies, all accomplished within narrow capillaries under high voltage (Figure 2). However, the type and medium of the capillary determines the strategy of the separation technique (16).

Figure 2: A schematic view of capillary electrophoresis (a) and several separation modes (b), including capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), micellar electrokinetic chromatography (MEEKC), and capillary electrochromatography (CEC). Reproduced from ref. (20). Capillary zone electrophoresis The principle of capillary zone electrophoresis (CZE) – the most common approach within CE – is based on the application of an electric field over a solution, which results in the migration of charged particles (16). The capillary is filled with a background electrolyte (BGE) also present in the electrode vials. After introduction of the sample via a small plug near the inlet vial, a voltage is applied. As a consequence, compounds are separated by charge-to-size ratio forming several zones with different velocities towards the detector. The time that the analyte reaches the detector is considered characteristic, whereas the peak height of the detection signal is related to the concentration of the analyte. The BGE – usually consisting of a buffered aqueous solution – serves as a buffer to maintain constant electrophoretic conditions throughout the analysis. During the analysis, several parameters highly influence the

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performance of CE, such as the pH of the BGE, ionic strength of the BGE, organic solvent added to the BGE, and interacting additives present in the BGE. Capillary gel electrophoresis Macromolecules with multiple charges – such as DNA fragments (21) – are unsuccessfully separated with CZE, since molecules of the same type but different size carry equal charge-to-size ratios. Therefore, CGE is developed wherein the capillary contains a polymer sieving gel allowing separation of charged compounds based on size. The smaller the compound, the faster it migrates, and the quicker it is detected. Adapting the type, size, and concentration of the sieving polymer, the selectivity of the separation can be optimized. The sieving gel commonly consists of chemically cross-linked polyacrylamide that forms a sieving network within the capillary (16). Though, liquid gels are also widely used in CGE containing for example linear polyacrylamides, polyvinyl alcohol, celluloses, and dextrans. The advantages of a liquid gel are that it has a relatively low viscosity which can be easily flushed through the capillary. A highly comparable technique is the so-called slab-gel electrophoresis approach. However, CGE allows a 100 times higher electric field resulting in an enhanced resolution. Additionally, it provides advantages of on-capillary detection, full automation, and easy use. Capillary isoelectric focusing CIEF is a CE approach which is based on the separation of mainly proteins with differences in isoelectric point (pI) (22). To facilitate such a separation, the capillary is carried out with a pH-gradient forming ampholytes. The positive electrode vial contains a solution with a low pH, whereas the negative electrode vial consists of a solution with a high pH. The analysis includes two procedures: focusing and detection, which can be performed in two ways. Focusing and detection can be applied in one sequence, in which an electroosmotic flow (EOF) is needed to focus the analytes and passing them through the detector. Another way is to first focus the analytes while maintaining a suppressed EOF. After mobilization of the separated zones, they are subsequently detected by either applying a hydrostatic pressure through the capillary or electrophoretically (16). The migration differences and thus the variance in pI is influenced by the protein diffusion coefficient, the electric field strength, the local pH gradient, and the mobility slope near the pH. Compared to gel isoelectric focusing (IEF), CIEF offers several advantages, such as a higher resolution due to the application of higher electric fields, on-capillary detection, full automation, and easy use. Micellar electrokinetic chromatography The main principle of MEKC is based on the separation of neutral compounds partitioning between the BGE and charged micelles (23). The capillary is filled with BGE containing surfactant allowing the formation of charged micelles when the concentration of the micelles reaches above the critical micellar concentration (CMC) (24). In contrast to the previous discussed CE modes, MEKC is a chromatographic instead of an electrophoretic separation technique. The BGE is considered the mobile phase, whereas the charged micelles serve as the stationary phase. Neutral compounds migrate between the EOF and the micelles. Influencing parameters are the type and concentration of surfactant, the pH of the BGE, ionic strength of the BGE, and the organic solvent added to the BGE. Capillary electrochromatography Another chromatographic CE approach is CEC, which is an electroseparation technique using an EOF to transport the mobile phase through a packed capillary (25). This in contrast to HPLC, since the latter is based on a flow driven by a pressure gradient. In CEC, separation of neutral compounds is allowed by a partitioning mechanism between the mobile and stationary phase,

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whereas charged compounds undergo both partitioning and differential electromigration. This technique is mostly applied for the separation of small neutral or weakly basic or acidic compounds. 2.2 Optical detectors Fluorescence detection in protein analysis One of the techniques allowing highly sensitive detection of small amounts of molecules down to sub-ppm quantities, is laser induced fluorescence (LIF) (26). LIF can be performed in both qualitative and quantitative analyses. When a fluorescent analyte has been exposed to laser light matching the transition energy of the molecule, the electrons within the chemical bonds will be transferred to a higher electronic state (27). Upon emission of the energy, the molecule emits light of a characteristic wavelength, which is called fluorescence. Since the excitation and emission wavelength are characteristic for the analyte, the detected fluorescence wavelength can yield information about the molecule’s identity. Additionally, the intensity of the emission light is proportional to the concentration of the analyte. In protein analysis, LIF is highly preferred since it features a high sensitivity. A drawback is that many proteins need to be labelled with fluorescent dyes, since many protein do not possess native fluorescence (28). UV-vis in protein analysis Another optical detection technique is UV-vis spectroscopy, wherein the molecule’s absorbance of monochromatic light is detected. In UV-vis spectroscopy the energy of the light beam falls within the UV-vis region, which usually ranges from the UV region to visible light (190 – 400 nm) (29). Using a reference light, the UV-vis absorbance of the analyte measured in a flow cell can be correlated to the concentration of the analyte. Typical analytes suitable for UV-vis detection contain unsaturated bonds, aromatic structures or functional groups including heteroatoms. For the analysis of proteins, absorption usually takes place at 280 nm(28). 2.3 Mass spectrometric detection Another detection technique widely used in this field, is mass spectrometry, wherein compounds are detected by mass-to-charge ratio (m/z) after being ionized (30). Once passed the separation device, the eluent will be analysed by MS containing four essential procedures: ionization, acceleration, mass analysis, and detection. During ionization, the eluent is eliminated and the compounds present in the sample will be charged; either because they were initially charged due to the used buffer or by ionization. Next, the ions will be accelerated through an electric field in order to give the ions energy and eliminate uncharged compounds. In the mass analyser, the ions are subsequently separated by m/z passing the ions through a magnetic field. The heavier the compound, the less it will be affected by the magnetic force, which results in a distinction between compounds based on mass. Finally, the ion beam will be detected resulting in a mass spectrum. Since ionization is essential for the identification procedure and highly influences the results obtained from the MS, two commonly used soft ionization techniques will be further explained: electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). Electrospray ionization (ESI) In ESI, the eluent coming from the separation device enters an ionization chamber at atmospheric pressure (Figure 3). The eluent passes through a needle carrying a high voltage, which results in an opposite migration of positive and negative ions to the needle or the counter electrode (31). Depending on the ionization mode – positive or negative – positive ions move to the end of the capillary forming a so-called Taylor cone containing an excessive positive

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charge at the surface of the eluent. The eluent surrounding the positive droplets present in the tip of the Taylor cone will be evaporated, which results in the formation of positive ions ready for further mass analysis. ESI is usually applied for the analysis of proteins or protein complexes. Matrix-assisted laser desorption/ionization (MALDI) The other commonly used ionization technique is MALDI, which is usually applied for the analysis of proteins, peptides and polymers. In MALDI, compounds in solution are mixed with a substance containing low molecular weight molecules and allowing absorbance of the chosen type of radiation, such as UV light (32). Thereafter, a vacuum is applied in order to evaporate the solvent from the matrix consisting of a crystal lattice in which the analyte is present. The matrix is subsequently irradiated with a laser of – for instance – UV light resulting in the desorption of the analyte from the matrix. The latter is followed by the ionization of the analyte, which allows further analysis of the analyte regarding their m/z.

Figure 3: Schematic view of the ESI setup. The eluent containing the analyte is passed through a needle with a high applied voltage. As a result, positive ions migrate to the tip of the needle forming a Taylor cone. The eluent is subsequently evaporated and positive ions remain. Ions are now ready to be analyzed by m/z. Reproduced from ref. (31).

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3. Intact biopharmaceutical analysis A more general term for co-existing forms of the biopharmaceutical, are isoforms. Isoforms include basically all possible variations of the same macromolecule. Since a mixed composition can affect the efficacy and safety of the product, the quality of a drug is influenced by the existence of isoforms. Determination of the biopharmaceutical’s concentration and the high molecular weight isoform (HMWI) relative ratios, enables manufacturers to criticize the quality of the production process (33). In this chapter we distinguish size variants and fragments of the biopharmaceutical, charge variants, and aggregates. 3.1 Size variants and fragments of the biopharmaceutical Kubota et al. aimed to identify a fragment of mAb-A, which is usually very challenging due to the low sensitivity regarding the small amount of the fragment (34). In order to allow degradation, mAb-A was subjected to a 6-months-during incubation time at 25 °C. For the separation, a BFS capillary was used accomplished with a SDS gel buffer running at 15 kV. Compounds were detected with UV at 220 nm. Once the fragment was separated by SDS-CGE (Figure 4), it was identified by in-gel digestion peptide mapping, RPLC-MS and subsequent fractionation device. As a result, they successfully identified the fragment as a fraction of heavy chain HC1-104, which influences the antigen binding activity and thus affects the efficacy of mAb-A. Additionally, they claimed that the amount of this fragment increases with 0.2 % a year when stored at 5 °C.

Figure 4: The electropherograms (a) of reduced SDS-CGE with in black the initial mAb-A and in red the degraded mAb-A. The reduced initial mAb-A shows an extra peak eluting before the heavy chain peak, whereas the reduced degraded mAb-A shows extra peaks coeluting with the internal standard and two peaks just before the elution of the heavy chain. The electropherograms of non-reduced SDS-CGE (b) shows a shoulder peak at the monomer. This indicates that the fragment is a fraction related to the heavy chain of the mAb.

Xu et al. have developed a CE two-antibody western blot setup to elucidate the titer and isoform distribution of proteins present in cell culture harvests and drug substance samples (33). In order to predict a drug sample’s composition, this method was compared to an SDS-PAGE method. The CE western was performed with Teflon-coated silica capillaries at 275 V on denatured and reduced samples. Analysis of the purified drug substance by SDS-PAGE resulted in the detection of four major polypeptide components, of which one assigned as a high molecular weight isoform, two smaller peptides due to intracellular enzymatic digestion of the HMWI, and one small peptide impurity. Except for the latter, these peptides were also detected in the CE western blot analysis. The obtained isoform distribution from CE western were used to generate a model for the HMWI percentage of the final drug substance. They succeeded in maintaining a good precision with a relative standard deviation of less than 10 % (RSD) as compared to the estimated 30 % RSD of conventional lab gel western blot. As

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described by the authors, one advantage of western blot over SDS-PAGE is the direct application of the method after harvesting cell culture samples instead of during batch release. Since SDS-PAGE did not lead to a sufficient resolution and peak symmetry for the separation of recombinant therapeutic protein-1 (RTP-1), Beckman et al. developed a CE setup wherein sodium hexadecyl sulfate (SHS) was added to the running buffer instead of SDS (35). They found out that the protein had low binding affinity with SDS and/or it failed to completely denature. Addition of SHS – and therefore longer alkyl chain detergents – instead of SDS has resulted in an enhanced resolution and higher plate counts. This buffer has been run at 15 kV through a capillary leading to a photodiode array detector. Introducing this method, they allowed characterization and release of RTP-1. In order to put CE performances in perspective, the article of Halim et al. shows us characterization analyses of biopharmaceuticals on different levels accomplished by four different techniques. SDS-PAGE, high-performance size exclusion chromatography (HP-SEC), AF4, and CZE were applied for the characterization of epoetin’s isoform distribution in different batches (36). Besides the observed differences between epoetin’s products, they found varying isoform distributions between batches of the same product. The analysis with non-reducing SDS-PAGE showed no HMWI or fragments in any of the tested product. Furthermore, HP-SEC showed clear monomer peaks for all batches, AF4 showed two or more peaks, and CZE detected six or more peaks. Despite the limitations of each technique, the products were still considered as high quality, since variations are inevitable due to the use of living cells throughout the production process. 3.2 Charge variants Another way to determine a product’s heterogeneity is by analyzing charge variants (37). Several modifications can result in charge variants of the reference product (38), such as C-terminal lysine variants, deamidation, formation of N-terminal pyroglutamate, glycation, and glycosylation (39) (37). The charge variants of mAbs are generally considered as acidic or basic variants, wherein acidic variants contain low pIs and basic variants high pIs (40). Since these variants can affect the immunogenicity and overall stability of biopharmaceuticals, monitoring the presence of charge variants is essential during the drug development process (38). Ahn et al. performed characterization analysis to the isoforms of recombinant human erythropoietin (EPO) (41). A CIEF-UV-vis setup included a capillary consisting of a hydroxypropyl cellulose (HPC) coating, an applied voltage of 25 kV, an HAc solution serving as the anode, and an ammonium hydroxide solution functioning as the cathode. Prior to CIEF, the samples were desalted with Amicon centrifugal filters. With this method, high resolution separation of seven isoforms were obtained, which was in agreement with previous CZE studies. The main advantage of this coating was the elimination of EOF without the need of additives, which makes it appropriate to couple with MS. Another interesting result was that the addition of 7 M Urea to the sample provided an enhanced separation due to the increased solubility of the proteins. Ahn et al. claimed that Urea prevents the formation of intermolecular hydrogen bonds which would lead to aggregation. Despite the preferences for non-denaturing buffers, adding urea to the buffer has resulted in enhanced separations of several mAbs (42). Henley et al. developed a promising Ultra-High Voltage CE (UHVCE) approach, whereby the separation resolution ideally increases with the square root of the applied voltage. The addition of 4 M urea to a buffer of 400 mM 6-

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aminohexanoic acid, 2 mM triethylene tetramine (TETA), and 0.05% hydroxypropylmethyl cellulose (HPMC) was essential for the enhancement of the separation. The HAc buffer was adjusted to a pH of 5.7 and the capillary was coated with PEG. Applying voltages up to 120 kV, they demonstrated an enormous improvement in resolution as well as decrease of analysis time compared to conventional CE setups (Figure 5). Where the latter shows two peaks in the UV-electropherogram (Figure 5a), the optimal UHVCE setup reveals two to three additional peaks (Figure 5d). Although there is still room for improvement regarding MS detection, they suggested to combine the PEG coated capillary with low conductivity buffers at UHV conditions in order to allow ESI-MS detection guaranteeing a fast separation with high peak capacity.

Figure 5: Electropherograms of four analyses applied to disulphide isomers.. The higher the applied voltage, the more isomers could be separated (from a to d). In order to keep the field strength consistent, the capillary length was increased when using higher applied voltages.

Redman et al. developed an intact microfluidic CE-ESI-MS approach in order to separate charge variants of an antibody drug conjugate (ADC) and to determine its drug-to-antibody ratio (DAR) (43). First, unconjugated IgG-2 was used leading to the CE separation of three main charge variants, which were further identified using the characteristic mass shifts from MS spectra and the electrophoretic mobility variations from the electropherograms. Using this method, they allowed separation of compounds which varied in mass of at least 18 Da. Second, conjugated IgG-2 have led to a reduced electrophoretic mobility and the separation of five species. These species – having a mass shift of 3145 Da – were considered as the mAb with 4 drug loads with an average DAR of 1.7. These results were comparable to the results obtained from infusion-ESI-MS and imaging CE of the same ADC. Bush et al. also used a CE-MS approach to conduct research to proteoforms of Avonex (44). This recombinant human interferon-!-1a was analysed on both intact level as well as in a top-down approach. A cross-linked polyethylenimine-coated bare-fused silica (BFS) capillary was used combined with a sheathless ESI interface connected to an Orbitrap MS. As a result, 138

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proteoforms were separated (Figure 6), of which 55 were quantified and 63 were identified by the corresponding deconvoluted MS1 spectra.

Figure 6: The ion density map of a CE-MS analysis of recombinant human interferon-β-1a. Besides the separation of 138 proteoforms, they allowed identification of 63 variants using the deconvoluted MS1 spectra. The remainder was identified based on their relative masses. Reproduced from ref. (44).

Another impressive way of separating intact monoclonal antibody variants was developed by Xiao et al., in which they created an open tubular capillary electrochromatographic (OT-CEC) method and created a fibrin-coated open column (Figure 7) (14). This technique was inspired by the natural phenomenon of blood coagulation. Using the thrombin-catalysed polymerization reaction, they were able to immobilize the highly cross-linked fibrin network onto the inner surface of a BFS column. The performance was tested with cetuximab, trastuzumab, and rituximab, and were compared to the analysis with a conventional BFS column. All analyses were operated with a BGE containing sodium phosphate, an applied voltage of 20 kV, and a UV-vis detector. Using this new strategy, the problem of positively charged mAbs adsorbed to the negatively charged capillary surface was overcome. Finally, the analysis has led to a good run-to-run and day-to-day RSDs of <1.64 % and a column-to-column RSD of < 2.42 %. Moreover, the fibrin-coated capillary allowed separation of 9 cetuximab variants and 5 rituximab variants, whereas the BFS capillary was limited to 7 and 4 variants respectively.

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Figure 7: Schematic setup of the complete procedure using OT-CEC for the characterization of mAb variants. Reproduced from ref. (14). Dai et al. introduced a high-resolution CIEF-MS method in order to separate charge variants of several intact mAbs, among which cetuximab (Figure 8) (45). This method allowed separation of three basic charge variants, one main peak, and five acidic variants. The setup contained an electrokinetically pumped sheath flow-nanospray ion source facilitating ionization prior to TOF analysis. Using a sheath liquid containing 10-30% ACN and 25-50% HAc, a sufficient MS sensitivity and robust mobilization were obtained. In order to generate high-resolution separation, 15-20% glycerol were added to the catholyte, samples, and anolytes were considered essential. Another factor affecting the resolution of the separation was the concentration of pharmalyte 3-10 in the buffer. A concentration of 1.5% was found to be optimal. Finally, introducing a urea-containing capillary rinsing step, the lifetime of the PS1-coated capillary and the repeatability of this method were improved. Faserl et al. investigated charge differences of several PTMs using a CE-MS setup (46). The latter consisted of a bare or neutrally-coated FS capillary, a BGE of 0.1 M HAc or 0.1% FA (v/v), and an applied voltage of 30 kV. Fragmentation of the peptides was realized by CID or HCD leading to the identification of the compounds. Based on the results, they claimed that this method allows separation of Asp and iso-Asp containing products caused by both in vitro and in vivo deamidation mechanisms. Furthermore, citrullinated proteoforms – which adds 0.984 Da to the mass and lowers the charge with 1 – were successfully separated.

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Figure 8: CIEF-MS (a), CIEF-UV (b) and CIEF-MS spectra (c) after separating charge variants of cetuximab. Both MS as well as UV detection have resulted in three basic variant signals, one main signal, and five acidic variant signals. Reproduced from ref. (45).

Hühner at al. have combined CIEF with MS using an additional CZE transfer loop applied on the separation of charge variants from a deglycosylated model antibody (47). Since the antibodies remain intact, this approach can be performed in the context of product stability testing. By using this two-dimensional method, they claimed to have solved issues, such as the ESI-interference of ampholytes obtained from CIEF, the close structure similarities of the variants, and the small volumes of CE-applications. In the arrangement of the CIEF-CZE-MS setup, the first-dimension CIEF separation was performed in combination with UV detection. A PVA-coated capillary was used to focus the mAbs by their pI values. After that, a switching valve introduced the analytes to the second dimension, wherein again a PVA capillary was used. The sheath liquid contained a solution of isopropanol:water (1:1) with 1 % HAc (v/v). The BGE consisted of 0.2 M FA. MS was carried out using a compact quadrupole-quadrupole time of flight (QqTOF) instrument. Upon separation of these charge variants, the first-dimension (CIEF) yielded four different signals in the electropherogram. Two of these peaks were further analysed by the second-dimension separation with MS using the multiple heart-cut procedure. A mass difference of only 2 Da and a significant shift in pI value have resulted in two different accurate masses (Figure 9). Although this setup allowed separation of these charge variants, additional techniques were necessary to ensure the occurrence of the charge difference-causing deamidation mechanism.

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Figure 9: The second-dimension results of the CIEF-CZE-MS analysis of the mAb X’s charge variants. A and B contain the cuts from the first-dimension signal 1 and 4 respectively. C and D are the corresponding MS spectra resulting in a mass difference of 2 Da between both mAb variants. Reproduced from ref.(47).

Another two-dimensional CE approach – developed by the same research group as the CIEF-CZE-MS method – showed similar results regarding the separation of charge variants of trastuzumab. Joos et al. used a comparable setup containing a CZE separation device in both dimensions (48). In the first dimension, 6 mg/mL trastuzumab was separated using a BGE containing 380 mM - ε-aminocaproic acid, 1.9 mM triethylenetetramine, and 0.05% (hydroxypropyl)methyl cellulose. During the analysis, they applied a voltage 10 kV on a BFS capillary. After the first-dimension separation, a UV detector was set at 214 nm in order to monitor the elution time of the mAb. A novel approach was used to bring the analyte in the right position within the separation loop to ensure the presence of the analyte prior to the second-dimension separation. Once the analyte reached the UV detector, the flow was stopped. Subsequently, a pressure of 50 mbar was applied to push the analyte back into the loop and properly position this analyte for the second-dimension CE analysis. In order to guarantee correct heart-cutting, they determined the time required at the low flow rate prior to each analysis. After switching the valve to the second-dimension CZE, the separation was accomplished using a BGE consisting of 2 M HAc. After separation through a PVA-coated capillary with an applied voltage of 10 kV, MS was carried out using a QqTOF device. The sheath liquid was composed of 1% HAc in 1:1 isopropanol-water. The results of this two-dimensional setup were promising, since they allowed separation of trastuzumab’s glycosylated and deglycosylated charge variants with standard deviations of less than 1 Da. Two out of four acidic charge variants, A2 and A3, were separated from the main peak with a mass difference of 2 Da and 3.1 Da respectively (48) (Figure 10). To allow these separations, an analyte concentration of 30 mg/mL was required in order to separate and detect A3. Nevertheless, considering the undiscussed charge variants A1 and A4, they were unable to

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determine the mass of all variants. In the context of PTM characterization, they stated that these mass shifts and migration times correspond to deamidiation products of the intact mAb.

Figure 10: The results of the CZE-CZE-MS approach analysing charge variants of trastuzumab. The UV chromatogram of the first dimension (a) shows one peak containg the basic variant, one peak of the main compound, and 4 peaks containing the four acidic charge variants (A1 to A4). A heart-cut volume of 10 nL was used in order to identify the main peak (b), whereas a volume of 20 nL was needed for the analysis of A2 (c). However, for the identification of A3, 20 nL sample was needed containing a 5-fold higher concentration. Reproduced from ref. (48).

An interesting combination of SDS-PAGE with reversed phase ultra performance liquid chromatography mass spectrometry (RP-UPLC-MS) was performed by Yamada et al., in which they analysed the isoforms of Rituximab based on differences in N-glycans (49). This elegant method allows analysis of intact mAbs, whereby no enzyme treatment is needed. After separation by SDS-PAGE, they introduced an extraction step using an SDC-containing buffer because of its solubilisation effect on proteins and the easy removal by organic solvents. RP-UPLC-MS was subsequently used in order to eliminate salts and detergents preventing ion suppression. Finally, this extraction step allowed a recovery of at least 86.4 (+/- 0.2 %) and resulted in the separation of four glycoforms differing in 162 Da. The latter is considered a galactose residue. Goyon et al. compared three chromatographic techniques separating ten different intact mAb’s using non-denaturing conditions (50). First, they performed SEC using a mobile phase consisting of 50 mM potassium phosphate and 250 mM potassium chloride in water (pH = 6.8). Samples with a concentration of 0.5 mg/mL were injected with a volume of 0.5 µL. Second, cation exchange chromatographic (CEX) was accomplished using a strong cation exchange column combined with mobile phase A containing 10 mM MES in water (pH = 6.0), and mobile phase B consisting of 10 mM MES and 1 M sodium chloride in water (pH 6.0). The following gradient was used: increase from 0 % to 20 % B in 20 min, increase from 20 % to 100 % B in 0.2 min, isocratic elution at 100 % B in 0.8 min, decrease from 100 % B to 0 % B in 0.2 min, and 8.8 min equilibration at 0 % B. This analysis was performed with an injection volume of 1.0 µL using the same concentration as in the SEC analyses. Finally, hydrophobic interaction chromatography (HIC) analyses were carried out using mobile phase A containing

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100 mM potassium phosphate and 2 M ammonium sulphate in water (pH = 6.8), and mobile phase B consisting of 100 mM potassium phosphate in water (pH = 6.8). The following gradient was applied: increase from 0 % to 100 % B in 40 min, isocratic elution at 100 % B for 5 min, decrease from 100 % to 0 % B in 0.2 min, and 4.8 min equilibration at 0 % B. The injection volume for the samples was 5.0 µL using the same concentrations as the other runs, except for brentuximab which had a concentration of 5 mg/mL. All analyses were performed with fluorescence detection set at an excitation wavelength of 280 nm and an emission wavelength of 340 nm. Throughout the study, Goyon et al. used generic instead of optimized conditions, meaning that the charge variants of the mAbs were not optimally separated (50). In general, it has been suggested that ion exchange chromatography (IEX) allowed the best separation for charge variants of mAbs, which is shown in figure 11. Although optimized separation conditions might have led to more successful results, the generic conditions already allowed a sufficient separation capacity. SEC analyses enabled separation of high and low molecular weight species from the main peak, although sometimes not with baseline resolution. Additionally, the chromatograms of all ten mAbs were highly similar, which means that they show an insufficient distinction. Furthermore, by using the HIC method they found that there is insufficient difference in hydrophobicity between the variants of the mAbs.

Figure 11: The separation of trastuzumab using three different techniques: SEC (a), IEX (b), and HIC (c). Reproduced from ref. (50).

Alekseychyk et al. proposed a CEX technique allowing rapid screening of charge variants of monoclonal antibodies (38). Anti-streptavidin type IgG1 and IgG2 – with pI values of 8.2 and 7.7 respectively – were analysed using acetate, citrate, and phosphate buffers as three different stress conditions. These samples were subjected to CEX analyses using three mobile phases: A, B, and C, consisting of 200 mM MES, 200 mM MES base, and 200 mM NaCl respectively. During the analysis, a salt gradient was set, and UV-absorbance was measured at 280 nm. Although the exact type of modification was unidentifiable, an additional data model allowed to properly determine the samples with the highest and lowest number of modifications. Besides that, the CEX chromatograms of both antibodies were rather different, resulting in a higher resolution for the IgG1 antibody. Another study to the charge variants of some mAbs using CEX, was performed by Wagner-Rousset et al. (40). Using both non-digesting as well as digesting conditions they allowed characterization of charge variants. Prior to the digested analyses, sample digestion was performed with IdeS. They used a strong cation exchange column with either a salt or pH gradient. The salt gradient consisted of a linear increase from 0 % to 20 % B in 20 min, and therefore, mobile phase A contained 10 mM MES in water, whereas mobile phase B contained

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10 mM MES and 1 M NaCl in water. Both buffers had a pH of 5.6 accomplished by the addition of 1 M NaOH. The CEX analyses with pH gradient were performed using a gradient with linear increase from 0 % to 100 % B in 30 min. Mobile phase A consisted of ten-fold diluted pH gradient buffer with pH 5.6, whereas mobile phase B was a ten-fold diluted pH gradient buffer with pH 10.2. Detection was carried out at an absorbance wavelength of 280 nm. Despite the successful characterization, identification of the acidic and basic variants remains a limitation. They suggest subjecting several fragments to LC-MS analyses, which is considered a time-consuming procedure. 3.3 Aggregates The problem of biopharmaceutical aggregation is clearly formulated by Bryan, who considers it as one of the causes of therapeutics becoming less effective after a certain period (51). Once proteins are produced within a living cell, they fold into a specific shape with hydrophobic parts turning towards the inner side and hydrophilic parts towards the outside. When proteins unfold – for example due to stress conditions – the inner part of the molecule can be exposed to the outside, which can lead to a complex formation due to self-association (52). These complexes are called aggregates and result in the loss of their therapeutic function and can affect the immunogenetic response. Storage conditions – with optimal and consistent environmental circumstances – are of great importance to prevent aggregation due to changed conditions in the packing. Therefore, analytical techniques to monitor the aggregation products and/or fragments at certain conditions are necessary.

Figure 12: SEC chromatograms of the four compared columns applied for of atumumab (A), natalizumab (B), brentuximab-vedotin (C) and trastuzumab-emtansine (D). Reproduced from ref. (53)

The separation of aggregates is mainly performed by other techniques than CE. One technique widely applied for the separation of aggregates is SEC (54). Goyon et al. aimed to compare the performance of four new commercially available sub-3 um SEC columns for the separation of mAbs and ADCs (53). Regarding the separation of aggregates, all columns allowed an average monomer-dimer resolution between 1.9 and 2.1. Figure 12 shows the chromatograms of the

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four tested columns applied on four mAbs. Additionally, they enabled an analysis time between 3 and 8 min. Between the performance of the different columns, they observed nonspecific interactions between the analyte and the packing of the columns, which was best prevented by using an AdvanceBioSEC column. Sahin et al. performed multiple strategies in order to determine protein aggregation of an influenza split-virus vaccine (55). This has resulted in a fast screening application for large aggregates allowing fast and reliable quality assessment. The following characterization techniques were combined: transmission electron microscopy (TEM), UV-vis spectroscopy, Nile Red fluorescence spectroscopy, HPLC, and single-radial immunodiffusion (SRID) assays. These multiple analyses have resulted in the detection of large protein aggregates partly caused by a certain surfactant present in the sample. Other non-CE approaches applied for the identification of aggregates are sedimentation velocity AUC (56), field-flow fractionation (FFF), and light scattering (54). Although SDS-PAGE was a widely applied technique in the past, other techniques are more popular nowadays. SDS-PAGE is still performed when applied for more in-depth characterization analysis, although not specifically for aggregates (49).

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4. Peptide and PTM analysis 4.1 N-linked glycan composition Glycosylation is the most common and complex PTM present in biopharmaceutical proteins (57) and it is estimated that about 50% of human proteins are glycosylated. Glycosylation is explained by the attachment of sugars to affect immunogenicity, solubility, protein folding, and serum half-life (4). This process affects the protein-protein interaction to support homeostasis (58). Manipulations in the glycosylation profile of specific proteins have been functioned as disease markers (6). Glycosylation leads to macro- and micro-heterogeneities in the core oligosaccharides and composition of the sugar chains. The composition of the sugar chains can range from single monosaccharides to complex linear oligo- or even polysaccharides affecting their biochemical and physiological activity (59). Glycosylation can be further classified into O- and N-linked variants among others. O-linked glycoproteins contain glycan chains attached to specific serine or threonine groups, whereas N-linked glycan chains are attached to asparagine (Figure 13) (60). In general, O-glycosylation is easier compared to N-glycosylation analysis, since the O-glycans are smaller and less branched carbohydrates (59). Furthermore, N-glycopeptides show more diversity in glycoforms. Considering the comparability studies of biopharmaceuticals required by the EMA, a complete profile of glycan structures contains the glycan pattern, site-specific glycosylation pattern and site occupancy (8). In this chapter, several approaches are described to elucidate the N-linked glycan structures.

Figure 13: Model glycoproteins illustrating the glycan chain positions. Whereas O-glycans contain glycan chains to specific serine or threonine groups, N-glycans have them attached to asparagine residues. Reproduced from ref. (61).

To evaluate N-glycan sequencing, Szigeti et al. developed a capillary electrophoresis laser-induced fluorescence (CE-LIF) method, which focused on the automation of a digestion-based sequence approach. Samples containing Immunoglobulin (IgG) and Enbrel N-glycans were

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separated using a BFS capillary, which functioned as a separation device and facilitated the enzymatic digestion reaction (62). In addition, the sample storage device was used as the temperature control. This method allowed successful digestion of the glycans and resulted in a complete N-glycan sequence in an analysis time of 60 min, and about 2 hours for the semi- and fully-automated approach respectively. In contrast, conventional CE methods need four days to achieve this. Kovacs et al. also developed a CGE-LIF method to analyse the presence of released N-glycans in serum from myeloma patients’ on both global as well as immunoglobulin level (63). These samples, also called para-proteins, may have a specific N-glycosylation profile that can serve as diagnostic indicators for multiple myeloma. For this purpose, they proposed a CE-LIF method compiled of NCHO capillaries with a NCHO separation gel buffer and applying 30 kV. The results showed differences between the global serum N-glycosylation profiles of normal control, untreated, treated and remission groups. However, on immunoglobulin level these differences were much less pronounced. Principal component analysis (PCA) applied to the total serum N-glycosylation yielded 12 N-glycans and showed significant differences between the disease stages, whereas the immunoglobulin analysis led to 6 identified N-glycans with similar significance. An extended approach of CE-LIF was developed by Feng et al. who worked on a system combining magnetic-bead sample preparation with multiple-capillary CGE separation and N-glycan profiling based on genetic analysis (64). In this study, 26 N-glycans were included of which were sialylated glycans, 11 non-sialylated glycans, and 10 glycans with high mannose contents. The whole sample preparation process was finished in 12 hours, whereas the subsequent CE separation was finished in only one hour. In the final step of the analysis – CGE separation by a DNA analyser – samples were injected from a well plate containing a labelled glycan solution and a loading mix which allowed parallel separation using a 16-capillary array and POP 7 polymer. Additional dye-labelling optimization showed that APTS labelling can overcome abundance problems experienced by other dyes. This labelling procedure contributed to a rapid and convenient glycan profiling approach, which is suitable for both high-throughput screening and profiling analysis as well as for cross-validation of N-glycans to allow peak identification (Figure 14).

Figure 14: The electropherogram of a CGE DNA analysis performed on 26 N-glycans. Peak assignment was realized by GeneMapper software which corresponded the RMU with reference glycan structures from a standard ladder. Reproduced from ref. (64).

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Giorgetti et al. proposed a method combining CE with ESI-MS in order to elucidate the structure and relative abundance of N-glycoproteins in therapeutic mAbs (65). In order to evaluate the CE-ESI-MS performance, the results were compared to similar analyses using hydrophilic interaction liquid chromatography fluorescence detection (HILIC-FD). Sheathless CE-ESI-MS was performed using a BFS capillary, a BGE consisting of 10 % HAc and an applied voltage of 20 kV. This method led to similar results for both CE-ESI-MS as well as HILIC-FD regarding the glyco-profile while guaranteeing high accuracy and precision. During an analysis time of less than 45 min, they achieved baseline separation for neutral and sialic acid glycans, including glycans differing in only one galactose. In general, HILIC analyses showed a very high precision with low standard deviations, whereas the suggested CE method contained low absolute variations of <4 %, similar to the performance of nanoLC-ESI-MS methods. Additionally, they found that both methods led to a similar glyco-profile based on the analysis of ten mAbs in triplicate. Guaranteeing high accuracy, precision, and robustness, using CE-ESI-MS they allowed characterization and quantification of both high as well as low abundant glycoforms. The study of Mancera-Arteu et al. focused more on the development of an efficient purifications step for O- and N-glycans and were analysed using CE-MS. CE-MS analysis was performed using a BFS column, a BGE containing 50 mM HAc and 50 mM FA (pH 2.2), and an applied voltage of 25 kV (59). The sheath liquid used for CE-MS consisted of isopropanol/water (50:50 v/v) with 0.05 % (v/v) of FA. Furthermore, the samples that were analysed included ovalbumin, human and bovine α1-acid glycoprotein, human apolipoprotein C-III, and recombinant human erythropoietin. Interestingly, they additionally studied the behaviour of O- and N-glycopeptides regarding the physicochemical parameters and characteristics of the compounds. This was relevant to obtain a broader understanding of the variables responsible for the compounds’ differences in behaviour during precipitation. Moreover, they observed that both the hydrophobicity as well as the size of the peptide chain have proven to play an important role in that trend. Kammeijer et al. invented a sheathless CE(-ESI)-MS approach suitable for separating differently sialylated glycopeptides (66). In this study, untreated %2,3- and %2,6-sialylated glycopeptide isomers were used. They combined a BFS capillary with a BGE consisting of 10% HAc (v/v) (pH 2.3), and an applied voltage of 20 kV. Development of the method was performed with %2,3- and %2,6-sialylated isoforms of IgG Fc, whereas the differences in electrophoretic mobility were again determined with %2,3- and %2,6-sialyllactose in terms of their pKa values. Finally, they applied the optimized method to tryptic glycopeptides of prostate specific antigen containing different sialic acid linkages. The latter has resulted in the separation of 75 forms of glycopeptides containing one single N-linked glycosylation site (Figure 15). Compared to a conventional CE-MALDI-TOF-MS setup, which enabled detection of only 37 N-glycans, the method developed in this study is very sensitive.

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Figure 15: CE-ESI-MS analysis of tryptic glycopeptides from PSA. The electropherogram of shows three clusters (A), of which the first contains the non-sialylated glycopeptides since they are the least acidic (B). The second cluster consists of mono-sialylated glycopeptides (C), whereas the last – and most acidic – di-sialylated glycopeptides are present in the third cluster (D). Reproduced from ref. (66)

Although glycan identification by MS might be challenging due to structure damage caused by ionization, Zhou et al. have overcome this problem by introducing a permethylation derivatization step. This method stabilises glycan structures prior to PGC-LC-MS/MS analysis and simplifies the MS spectra (67). In addition, the MS/MS setup is highly advanced in a way that it shows orthogonal fragmentation patterns between conventional collision-induced dissociation (CID) MS/MS and with higher-energy collisional dissociation (HCD) MS/MS providing different fragmentation patterns. In this study, the authors succeeded in separating and identifying glycan isomers with different fucosylation sites and galactose linkages. Moreover, the loss of monosaccharides during ionization was eliminated introducing the permethylation derivatization step and resulted in the assignment of monosaccharides to specific locations within the glycans. In the context of comparability studies between a biopharmaceutical and its biosimilar, Montacir et al. used a middle-up approach to elucidate the mass of different parts of rituximab and a biosimilar (8). To this end, limited proteolysis was performed to fragmentize between the Fab and Fc region and dithiothreitol (DTT) treatment was applied to reduce disulphide bonds. Subsequent UHPLC-QTOF-MS analysis led to glycoform assignments within the Fc/2 part of both products with slight mass increments (Figure 16). The same applies more or less for the LC and Fd fragments of Rituximab and the biosimilar. These results were confirmed by using the orthogonal denaturing ESI-QTOF analysis. Further glycopeptide analyses, to

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elucidate the glycosylation site(s) and occupancy, were performed using two approaches on intact and subunit level after tryptic digestion: UHPLC-QTOF-MS and MALDI-TOF-MS. In conclusion, both products showed similar glycosylation profiles with different quantities.

Figure 16: UHPLC-QTOF-MS results of the Fc, LC, and Fd mAb fragments after limited proteolysis digestion of rituximab (A, B, and C) and the biosimilar (B, D, and F). Reproduced from ref. (8)

Falck et al. performed glycosylation analysis on EPO present in cell culture supernatant using a HILIC-SPE and MALDI-TOF-MS approach (68). The analysis time covering from EPO purification up till glycan profiling was accomplished in about 24 hours. Additionally, they compared the glycosylation profiles obtained from this novel setup to a conventional CGE-LIF method. In regard to N-glycosylation, this method allowed high repeatability profiling while maintaining a coefficient of variation (CV) between 2.3 % and 6.2 % for major glycans and between 3.5 % and 20.2 % for minor glycans. However, the method still needs improvement on the location distinction between N-acetyllactosamine units, which is also essential in the quality control of pharmaceuticals. 4.2 Glycosylation site ID and O-linked analysis Although N-glycan analytical methods are readily developed, O-glycan analysis – consisting of the release, labelling, separation, and detection of O-glycans (58) – is more challenging due to the lack of O-glycan-specific enzymes (69). In today’s analytical techniques, non-enzymatic release of O-glycans are used, such as !-elimination. A more general approach to characterize glycoproteins, and not specifically for N- or O-glycoproteins, is developed by Engel et al. (70). Prior to the analysis of glycoproteins using microchip capillary gel electrophoresis (MCGE), lectin enrichment was carried out. The aim of this research was to find a new approach allowing glycoprotein analysis extracted from complex samples, which makes a successful glycoprotein enrichment step essential. To this end, they used samples containing different kind of glycosylated proteins as well as a non-glycosylated negative control. During glycoprotein enrichment, glycoproteins were first labelled with a fluorescent dye enabling LIF detection. Parallel to that, magnetic beads were prepared by coating covalently-bound lectins to the bead surface. Glycoproteins were

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subsequently captured based on their trimannosidic core structure present in N-glycoproteins, and other high-mannose structures in the glycoproteins. Elution of the glycoproteins was carried out adding competitive mono- and disaccharides which allows further characterization analysis of the glycoproteins. After enrichment of the glycoproteins, MCGE was applied containing the following procedures (70). After labelling the samples with a HSP-250 fluorescent dye, samples were diluted in water and subsequently denaturised with sample buffer containing DTT. Another procedure was performed using a P230 assay in which labelling was not necessary and samples were only diluted in water. The performance of the MCGE approach was compared to the analysis with SDS-PAGE. Upon comparison, they found similar results and additionally some benefits for MCGE: small sample volumes and shorter analysis times. When analysing complex mixtures, MCGE analysis still showed good selectivity. Although this technique showed some very promising features, it has some limitations such as the occurrence of unspecific interactions between the sample analytes and the magnetic beads during the analysis of complex mixtures. Kim et al. developed a nanoLC-Chip-Q/TOF MS technique for characterization purposes of O-glycosylated recombinant human erythropoietins (rhEPOs) (69). This included the profiling of bioactive sialic acid and O-acetyl side-chains. This approach successfully elucidated structural information regarding the O-glycan composition, the O-glycosylation site, and the location of O-acetylation. In order to characterize the O-glycan structures, they performed a glycoproteomic approach composed of ultrafiltration, digestion, SPE purification, nanoLC separation, and (tandem) MS (69). Prior to that, 50 µgglycoproteins were dissolved in an alkaline mixture consisting of 1.0 M NaBH4 and 0.1 M NaOH. Afterwards, ultrafiltration was performed using membranes with a MWCO of 10 kDa to eliminate excipients that possibly interfere throughout the analysis. In-solution digestion was carried out with a non-specific protease pronase E in a phosphate buffer of pH 7.5, which transformed rhEPO into O-glycopeptides. For the enrichment of the O-glycopeptides, a porous graphitized carbon (PGC) SPE-cartridge was used wherein elution of the sample was performed with a solution of 40 % ACN and 0.05% (v/v) TFA in water. The same separation mechanism, namely PGC, was applied on the nanoLC chip-Q/TOF MS and tandem MS analysis. Mobile phase A consisted 3.0 % ACN and 0.5 % FA (v/v) in water, whereas mobile phase B contained 90 % ACN and 0.5 % FA (v/v) in water. The gradient showed a linear increase from 5 to 35.9 % B in 16.5 min. Prior to MS detection, ionization was carried out by nano-ESI. The nanoLC-PGC chip-Q/TOF MS approach have led to the identification of 9 glycopeptides in the first generation rhEPOs and 19 glycopeptides in the second generation (69). The offered technique allowed not only separation and identification of O-glycopeptides containing different amounts of O-glycans, but also separation of mono- and di-sialylated O-glycopeptides. Furthermore, they were able to make a distinction between high and low O-acetylated glycopeptides containing sialic acids. Among the mono-sialylated O-glycans, two isomers were successfully separated, which makes this method also suitable for isomeric identification. One drawback in the identification of O-glycans, is the inability of O-acetylated compound detection at harsh alkaline conditions. A given explanation is that these conditions resulted in the destruction of chemical labile bioactive glycans, such as sialylation and O-acetylation. Tandem MS analysis have led to the elucidation of the O-acetyl-modification site and the O-glycan structure (69). Specific fragment losses have led to the determination of certain parts of the compound, such as NeuAc residues. Moreover, specific fragment ions can be used in order to find the O-glycosylation site (126Ser).

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A comparable method was applied by Seo et al. from the same research group (71). Their aim was to investigate the micro-heterogeneity and macro-heterogeneity within the O-glycosylation structure of a recombinant coagulation factor IX (rFIX) necessary to understand its pharmacological activity. To achieve this, the following method was set up. First, they removed detergents with an ultrafiltration membrane with a molecular weight cut-off (MWCO) of 10 kDa. Subsequently, they treated rFIX with a solution of 1.0 M sodium borohydride and 0.1 M NaOH in order to release the O-glycans. After enzymatic digestion, another ultrafiltration step was performed in order to remove N-glycans and digestion chemicals. During the subsequent PGC-SPE enrichment step, similar conditions were used as described in the study of Kim et al., except for the elution of the O-glycans (69) (71), whereby stepwise elution starting with 20 % ACN in water and 40 % ACN in water containing 0.05 % (v/v) TFA was performed (71). During the gradient nanoLC-analysis, mobile phase A consisted of 3.0 % ACN and 0.5 % (v/v) FA in water, whereas mobile phase B contained 90.0% ACN and 0.5 % (v/v) in water. Different gradients for the elution of O-glycans were used compared to the elution of O-glycoproteins, as the latter elutes at higher concentrations of organic modifier. The ionization for MS analysis was again carried out by nano-ESI. Furuki et al. mainly focused on the release of the O-glycans using a new approach called RapiFluor-MS labelling combined with non-reductive β-elimination and LC-MS/MS (58). Prior to that, non-enzyme sample treatment was performed on reduced O-glycans present in glycoproteins. RP-ESI-MS/MS was accomplished using this technique and contributed to a successful structure analysis of both major as well as low-abundance O-glycans in cytotoxic T lymphocyte associated protein 4-immunoglobulin G (Ig) fusion protein (CTLA4-Ig) and fetuin. Additionally, a high sensitivity was obtained. This method allowed Seo et al. to identify six O-glycosylation sites containing uncommon O-fucosylations and O-glucosylations positioned at 53Ser and 61Ser (71) among others. The PGC stationary phase, allowing separation of compounds based on size and polarity, resulted in clear separation of each O-glycan. This chromatographic strategy has the ability to distinguish neutral O-glycans (no sialic acid residues) from the acidic O-glycans with at least one sialic acid residue. Each O-glycosylation was further categorized into O-glycosylation, O-fucosylation, and the common O-glycosylation containing a core 1. Quantitation analysis was performed to determine the peak areas from the extracted compound chromatograms, which could be related to the relative abundance of each O-glycan within the rFIX samples. Comparable to the study of Kim et al., the location of the O-glycans was successfully determined by CID MS/MS analysis.

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5. Amino acid sequencing Another quality assessment for monitoring biopharmaceuticals’ manufacturing processes includes the mapping of the amino acid sequence. The latter provides information about the building blocks of the protein, which are one of the responsible factors of the drug’s stability and thus the shelf-life (72). Therefore, amino acid analysis of biopharmaceuticals is another essential step within quality assessment.

Figure 17: The base-peak electropherogram (BPE) and extracted-ion electropherogram (EIE) of two ions detected in the CE-MS/MS analysis of TRI-1144. In both ions seven peaks could be distinguished corresponding to the native, singly (2 peaks), doubly (2 peaks), triply and quadruply-deamidated forms. Each of these degradation forms had an additional deacetylated form corresponding to the other 7 peaks. Reproduced from ref. (73)

Dominguez-Vega et al. tested a new CE-MS/MS approach to analyze the composition and site-specific deamidation profile of the degraded peptide therapeutic TRI-1144 (73). A BFS capillary was polybrene-dextran sulfate (PB-DS) coated with a solution of 10 % (w/v) PB and 3 % (w/v) DS and resulted in a significant pH-independent EOF, which contributes to an analysis with high efficiency and reproducibility. The optimized BGE consisted of 150 mM ammonium formate (pH 6.0)/ACN/isopropanol (60:35:5 v/v/v). To allow tandem MS analysis, the sheath liquid was composed of isopropanol/water/FA (50:50:0.1 v/v/v). This method allowed successful characterization of the amino acid sequence and PTM determination of TRI 1144, whereby the theoretically correct sequence with four deamidation and deacetylation sites was determined (Figure 17). MS has proven to be essential for the distinction of these two

33

PTM’s since the difference is only 42.011 Da. Moreover, tandem MS even allowed separation between deamidated products with a difference less than 0.984 Da. As a part of the study of Miao et al. to completely characterize tocilizumab, the amino sequence of this mAb and its biosimilar called HS628 were studied (7). To this end, they performed reduced tryptic peptide mapping using a RP-UPLC-QTOF setup with additional UV detection. The amino sequences of both tocilizumab as well as HS628 were compared and resulted in a 100 % match in their peptide profiles and the sequences were similar to the theoretical sequence known from the manufacturer. As discussed earlier, the amino acid sequence can still be similar among biosimilars while behaving differently, which is confirmed in this study. A similar approach regarding amino acid sequencing was used by Mistarz et al., in which they characterized a potential chimeric malaria vaccine, called GMZ2’.10C by using LC-MS/MS (74). For peptide mapping, tryptic digests of GMZ2’.10C was analyzed on an RP pre- and analytical column and detected with an ESI-QTOF mass spectrometer. The peptide map has resulted in a match of 88.3% with the compared to the sequence obtained from literature. Another study by Pisupati et al. also performed amino acid sequencing by peptide mapping using a reversed phase nanoUPLC-MS/MS after trypsin digest (75). In this study, they compared multiple characteristics between highly similar mAbs remicade and remsima, among which their amino acid sequence and PTMs. As a result, they found a >98 % match in sequence and equal amounts of oxidation and deamidation sites. Montacir et al. performed amino acid peptide mapping and identification of PTMs using the bottom-up approach, in which LC-ESI-MS/MS was performed on rituximab and a biosimilar (8). As a result, they found very high similarities between PTM types and modified peptides of both products. Analyses of the PTMs showed that only oxidation and deamidation were partially present in both products. In general, both products contained similar numbers and locations of PTMs.

34

6. Discussion Research to the protein’s identity and major isoforms is accomplished in several ways, varying from CE approaches, to SEC and AF4 methods. However, in general, separation based on charge has an added value to this type of analysis, since isoforms mainly differ in charge rather than size. Consequently, the size-based separation techniques AF4 and SEC showed less peaks compared to CZE when performed on the same samples (36). Although SDS-PAGE is a commonly used technique regarding isoform and/or fragment separations, it becomes impractical when applied to complex matrices and low analyte concentrations. CIEF’s resolving power – based on differences in pI – is indispensable and thus offers a high resolution regarding the separation of isoforms (41). The problem with CIEF is that the EOF must be eliminated in order to acquire stable zones. The CIEF-UV-vis strategy containing HPC-coated capillaries has overcome this problem. However, coupling with MS is not possible yet. In contrast, the advantages of both electrophoresis and MS are combined in the SDS-PAGE setup with subsequent RP-UPLC-MS analysis (49). This elegant technique allows analysis on intact mass level and therefore direct determination of isoforms at protein level. Besides no treatment was needed, the isoforms can be identified by MS instead of UV, which makes this strategy even more valuable. For the analysis of charge variants several approaches have been proposed during the last two years. CE-MS has been enormously successful, since this technique allowed separation of 138 charge variants (44). Within the CIEF method, both MS and UV detection were used leading to the same number of detected charge variants. However, using the CIEF method, ampholytes might interfere during ESI, which seems to be overcome performing the two dimensional CIEF-CZE-MS. Since the detection method in the first dimension is UV, one needs a reference ladder in order to select the right peak in the first dimension (47). A very successful feature of this technique is the separation capacity, which allows separation of compounds differing in only 2 Da. These results are comparable with the slightly different setup of CZE-CZE-MS, which is also a very elegant way of separating charge variants (48). A limitation of this technique is that it is unable to detect all variants present in the sample. Another CE approach applied is OT-CEC containing a fibrin network on the inner capillary wall. With this method, the problem of positively charged compounds adsorbing to the capillary wall is overcome (14). Overall, CIEF-MS, two-dimensional CE-MS combinations, and IEX show comparable results. SEC and HIC approaches were considerably less able to distinguish charge variants, which is explainable. Since charge variant mainly differ in charge rather than size, SEC does not allow distinction between the modified mAbs (50). Additionally, HIC seemed to be only suitable when the charge variants have enough diversity in hydrophobicity, which makes the CE and IEX approaches more robust. An alternative technique for the separation of isoforms is CE two-antibody-western blot (33), however, it requires laborious manual operations and often has an insufficient accuracy and precision. Interestingly, the two-dimensional CE-western-blot strategy has overcome the biggest part of these problems, since it makes use of photochemical capture of the analyte into the inner capillary wall in the first dimension. As a result, this technique is very valuable regarding complex samples. Additionally, comparing western blot to SDS-PAGE, the main advantage of western blot is the application directly after harvesting cell culture instead of during batch release. In that way, the quality of the production process can be earlier adjusted which is more convenient. Several CE approaches are particularly suitable for N-linked glycan analysis. CE has been coupled to both LIF and MS detection, which have led to complete glycosylation profiles.

35

Although CE-LIF is a rapid technique guaranteeing high resolution, high separation selectivity, and efficiency, the limitations of using LIF is the need to label the glycans and the use of a reference glycan ladder to identify the glycans. The latter means that only known glycan structures can be identified. CE-MSn has the benefits of both electrophoretic separation and bottom-up peptide mapping (66). Moreover, it shows high accuracy, precision and robustness combined with a short analysis time and the need of small sample volumes. In contrast to the conventional bottom-up approach (LC-MSn), in CE-MSn low-abundant glycopeptides are more easily detected and closely related peptides are separated based on charge. The approach of HILIC-SPE and subsequent MALDI-TOF analysis has the advantage of allowing high throughput analyses and obtaining high information content (68), although the SPE might be less able to separate compounds when applied to impure complex mixtures. Much less research is conducted specifically to O-linked glycans, compared to N-glycans analysis. Comparing MCGE to SDS-PAGE (70), MCGE requires smaller sample volumes, it needs shorter analysis times, and it still shows a good selectivity when applied to complex mixtures. Nevertheless, the use of this technique in combination with the magnetic bead purification step can still be improved due to the unspecific analyte-bead interactions. Besides MCGE, LC methods containing PGC-packed stationary phases are commonly used methods leading to successful O-glycan analysis (58). Besides the possibility to detect analytes with MS, no labeling techniques are required. Over the last two years, separation of aggregates on intact protein level is performed using SEC, FFF, and AUC, rather than electrophoretic methods. Although SDS-PAGE – another technique suitable for separation of aggregates – was widely applied in the past, other techniques have gained more interest due to shorter analysis times or better performances in general. For instance, SEC runs were finished in only 3 to 8 minutes and offer a resolution between 1.9 and 2.1 (53). Regarding amino acid sequence analysis, the commonly applied technique is LC-MS rather than CE methods. Reduced and non-reduced peptide mapping strategies – such as bottom-up and top-down approaches – are popular in order to elucidate the primary sequence of the product. However, the limitation of RPLC-MS is that structurally closely related peptides underwent PTMs, might be difficult to separate because this method lacks the resolving power based on charge (73). CE approaches might be more valuable when combining several characterization purposes, such as amino acid sequencing, PTM analysis and separating isoforms. Additionally, it allows separation of intact peptide therapeutics prior to MS, which reduces the complexity of mass spectra. However, when analyzing only the amino acid sequence of a certain compound, LC-MSn seems more convenient due to the efficient bottom-up and top-down approaches towards peptide mapping.

36

7. Conclusion Nowadays, CE have been developed to a certain extent allowing complete characterization of biopharmaceuticals. For the analysis of isoforms several CE approaches have grown to a highly-advanced method, which have resulted in more successful results compared to chromatographic techniques. The same applies to the characterization of charge variants, N-linked, and O-linked glycan analysis. For analyses regarding aggregation products and amino acid sequence, other methods have proven to yield more in-depth information and resolving power. In general, CE has become a very powerful technique in the field of biopharma, since many PTMs are related to changes in electrophoretic mobility. One suggestion regarding O-glycosylation analysis, is to conduct more analysis using CE approaches. At the moment, O-glycan structure analysis is hampered by the lack of enzymatic digestion techniques, which makes it difficult to apply on low concentrated samples. In addition, two dimensional CE approaches offer a convenient outcome for elimination of ion-suppressing compounds, which is a promising strategy for the future. The principle of sample extraction from the first dimension into the second dimension was also applied using SDS-PAGE and UPLC-MS.

37

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Appendix I: Overview methods applied regarding characterization analysis during the last two years Intact biopharmaceutical analysis

Mode Compound BGE composition / running buffer / eluent

Capillary medium

Applied voltage

Detector / ionization settings Reference

SDS-CGE and RPLC-MS

mAb-A SDS gel buffer BFS 15 kV UV at 220 nm and 214 nm ESI-QTOF

(34)

Western Blot Cell culture harvests and drug substance

- Teflon-coated silica

275 V CCD camera (33)

SHS-PAGE recombinant therapeutic protein-1

SHS buffer - 15 kV UV (35)

SDS-PAGE Epoetin 15% polyacrylamide gel with Tris-HCl pH 6.8, glycerol, sodium dodecyl sulfate and bromophenol blue

- 150 V Silver staining (36)

SEC Epoetin 14.4 g/L Na2HPO4.2H2O, 0.2 g/L KH2PO4, and 23.4 g/L NaCl (pH 7.4)

- - UV at 280 nm (36)

AF4 Epoetin 14.4 g/L Na2HPO4.2H2O, 0.2 g/L KH2PO4, and 23.4 g/L NaCl (pH 7.4)

- - MALLS and UV at 280 nm (36)

CZE Epoetin 0.01 M tricine, 0.01-M sodium chloride, 0.01 M sodium acetate, 7 M urea, and 25 mM putrescine (pH 5.55), adjusted with 50% (v/v) glacial acetic acid

BFS Field strength: 143 V/cm

UV at 214 nm (36)

CE-MS IgG-2 10% 2-propanol 0.2% HAc in water (pH 3.17)

NHS-PEG coating

35 kV TOF (43)

CE-MS Avonex (recombinant human interferon-β-1a)

3 % HAc in water (pH 2.5) cross-linked polyethylenimine-coated BFS

20 kV Orbitrap ESI-voltage: 1.6 kV

(44)

OT-CEC Cetuximab, Trastuzumab, and Rituximab

Sodium phosphate buffer Fibrin-coated BFS

2.1 kV UV-vis at 214 nm (14)

CIEF-MS mAbs Catholyte: 0.2 M NH4OH with 15% glycerol Anolyte: 1% FA with 15% glycerol

Neutral coating PS1

Field strength: 250 V/cm

ESI-TOF ESI-voltage: 2.0-2.4 kV

(45)

CE-MS Asp and iso-Asp containing products

0.1 M HAc or 0.1% FA Bare or neutrally FS

30 kV CID or HCD (46)

43

CIEF-CZE-MS deglycosylated model antibody

0.2 M FA in water PVA-coated 10 kV ESI-QqTOF Sheath liquid: isopropanol:water (1:1) with (v/v) HAc

(47)

CZE-CZE-MS Trastuzum 1st dimension CZE: 380 mM - ε-aminocaproic acid, 1.9 mM triethylenetetramine, and 0.05% (hydroxypropyl)methyl cellulose 2nd dimension CZE: 2 M HAc

BFS and PVA-coated

10 kV UV at 214 nm Sheath liquid: 1 % HAc in isopropanol-water (1:1, v/v)

(48)

HIC mAbs Mobile phase A: 100 mM potassium phosphate and 2 M ammonium sulphate in water (pH = 6.8) Mobile phase B: 100 mM potassium phosphate in water (pH = 6.8)

- - FD at (ex: 280 nm/em: 340 nm) and UV from 220 – 320 nm

(50)

SEC mAbs 50 mM potassium phosphate and 250 mM potassium chloride in water (pH = 6.8)

- - FD at (ex: 280 nm/em: 340 nm) and UV from 220 – 320 nm

(50)

IEX mAbs Mobile phase A: 10 mM MES in MQ (pH 6.0) Mobile phase B: 10 mM MES and 1 M NaCl in MQ (pH 6.0)

- - FD at (ex: 280 nm/em: 340 nm) and UV from 220 – 320 nm

(50)

CEX mAbs 200 mM MES, 200 mM MES base, and 200 mM NaCl

- - UV at 280 nm (38)

CEX mAbs Mobile phase A: 10 mM MES in water Mobile phase B: 10 mM MES and 1 M NaCl in water

- - UV at 280 nm (40)

TEM, HPLC, UV-vis, fluorescence, SRID

Split-virus vaccine - - - Uv-vis, Nile Red fluorescence (55)

CEX mAbs Mobile phase A: 10 mM MES in water (pH = 6.0) Mobile phase B: 10 mM MES and 1 M sodium chloride in water (pH 6.0)

- - FD at (ex: 280 nm/em: 340 nm) (53)

44

Peptide and PTM analyses Mode Compound BGE composition / running buffer /

eluent Capillary medium

Applied voltage

Detector / ionization settings Reference

CGE-LIF IgG and Enbrel - BFS 30 kV LIF (ex: 488 nm/em: 520 nm) (62) CGE-LIF Para-proteins in

serum NCHO gel NCHO 30 kV LIF (ex: 488 nm/em: 520 nm) (63)

CGE-LIF Mix of 26 N-glycans POP 7 polymer - - LIF (64) CE-MS mAbs 10 % acetic acid BFS 20 kV ESI-TOF

ESI-voltage: 1.75 kV Sheathless

(65)

CE-MS Ovalbumin, human and bovine α1-acid glycoprotein, human apolipoprotein C-III, and recombinant human erythropoietin

50 mM HAc and 50 mM HFor (pH 2.2) BFS 25 kV ESI-TOF Capillary voltage: 4 kV Sheath liquid: iPrOH/water (50:50 v/v) with 0.05 % (v/v) of HFor

(59)

CE-MS α2,3- and α2,6-sialylated glycopeptide isomers

10% AA (v/v) (pH 2.3) BFS 20 kV ESI (66)

LC-MS/MS Glycan structures Mobile phase A: H2O:FA (99.9:0.1, v/v) Mobile phase B: ACN:H2O:FA (98:2:0.1 v/v/v)

PGC - ESI-Orbitrap ESI-voltage: 1.6 kV

(67)

LC-MS/MS Rituximab and biosimilar

- - - ESI (8)

SPE-MS EPO - HILIC (SPE) - MALDI-TOF Acceleration voltage: 25 kV

(68)

Microchip CGE Glycoproteins from serum

- - - LIF (70)

nanoLC-Chip-MS Recombinant human EPO

Mobile phase A: H2O:ACN:FA (96.5:3:0.5, v/v/v) Mobile phase B: ACN:H2O:FA (90:9.5:0.5, v/v/v)

PGC - nanoESI-QTOF (69)

nanoLC-Chip-MS Recombinant coagulation factor IX

Mobile phase A: H2O:ACN:FA (96.5:3:0.5, v/v/v) Mobile phase B: ACN:H2O:FA (90:9.5:0.5, v/v/v)

PGC - nanoESI-QTOF (71)

UPLC-MS CTLA4-Ig and fetuin Mobile phase A: H2O:TFA (99.9:0.05, v/v) PGC - IT-TOF ESI

(58)

45

Mobile phase B: ACN:H2O:TFA (80:19.9:0.05 v/v/v)

Capillary voltage: 3.0 kV

Amino acid sequencing

Mode Compound BGE composition / running buffer / eluent

Capillary medium

Applied voltage

Detector / ionization settings Reference

CE-MS/MS Peptide therapeutic TRI-1144

150 mM ammonium formate (pH 6.0)/ACN/Isopropanol (60:35:5 v/v/v)

PB-DS-coated BFS

30 kV Sheath liquid: isopropanol/water/PR (50:50:0.1 v/v/v)

(73)

RP-UPLC-MS Tocilizumab and biosimilar HS628

Mobile phase A: H2O:TFA (99.9:0.1, v/v) Mobile phase B: ACN:TFA (99.9:0.1, v/v)

BEH C18 - UV at 214 nm Q-TOF: Capillary voltage: 3.0 kV

(7)

LC-MS/MS Malaria vaccine Mobile phase A: H2O:FA (99.7:0.23, v/v) Mobile phase B: ACN:FA (99.7:0.23, v/v)

BEH C18 - ESI-Q-TOF (74)

LC-MS/MS Rituximab and biosimilar

- - - ESI (8)