Glycoproteomics of a Single Protein: Revealing Tens of Thousands of Myozyme Glycoforms by Hybrid HPLC-MS Approaches.

Characterization of highly glycosylated biopharma-ceuticals by mass spectrometry is challenging because of the huge chemical space of coexistent glycoforms present. Here, we report the use of an array of HPLC-mass spectrometry-based approaches at different structural levels of released glycan, glycopeptide, and hitherto unexplored intact glycoforms to scrutinize the biopharmaceutical Myozyme, containing the highly complex lysosomal enzyme recombinant acid α-glucosidase. The intrinsic heterogeneity of recombinant acid α-glucosidase glycoforms was unraveled using a novel strong anion exchange HPLC-mass spectrometry approach involving a pH-gradient of volatile buffers to facilitate chromatographic separation of glycoforms based on their degree of sialylation, followed by the acquisition of native mass spectra in an Orbitrap mass spectrometer. Upon considering the structures of 60 different glycans attached to seven glycosylation sites in the intact protein, the large set of interdependent data acquired at different structural levels was integrated using a set of bioinformatic tools and allowed the annotation of intact glycoforms unraveling more than 1,000,000 putative intact glycoforms. Detectable isoforms also included several mannose-6-phosphate variants, which are essential for directing the drug toward its target, the lysosomes. Finally, for the first time, we sought to validate the intact glycoform annotations by integrating experimental data on the enzymatically dissected proteoforms, which reduced the number of glycoforms supported by experimental evidence to 42,104. The latter verification clearly revealed the strengths but also intrinsic limitations of this approach for fully characterizing such highly complex glycoproteins by mass spectrometry.

3D-LC-MS with 2D Multimethod Option for Fully Automated Assessment of Multiple Attributes of Monoclonal Antibodies Directly from Cell Culture Supernatants.

Fully automated analysis of multiple structural attributes of monoclonal antibodies (mAbs) using three-dimensional liquid chromatography-mass spectrometry (3D-LC-MS) is described. The analyzer combines Protein A affinity chromatography in the first dimension (1D) with a multimethod option in the second dimension (2D) (choice between size exclusion (SEC), cation exchange (CEX), and hydrophobic interaction chromatography (HIC)) and desalting SEC-MS in the third dimension (3D). This innovative 3D-LC-MS setup allows simultaneous and sequential assessment of mAb titer, size/charge/hydrophobic variants, molecular weight (MW), amino acid (AA) sequence, and post-translational modifications (PTMs) directly from cell culture supernatants. The reported methodology that finds multiple uses throughout the biopharmaceutical development trajectory was successfully challenged by the analysis of different trastuzumab and tocilizumab samples originating from biosimilar development programs.

Similarity demonstrated between isolated charge variants of MB02, a biosimilar of bevacizumab, and Avastin® following extended physicochemical and functional characterization.

The majority of recombinant mAb products contain heterogeneous charge variants, commonly the result of post-translational modifications occurring during cell culture and accumulated during production, formulation and storage. MB02 is a biosimilar mAb to bevacizumab. Similarity data of charge variants for biosimilars against its reference products must be generated to demonstrate consistency in product quality and to ensure efficacy and safety. The goal of this work was to isolate seven charge variants of MB02 and Avastin® by semi-preparative cation exchange chromatography followed by purity test and extended analytical characterization to prove similarity. Although poor purity obtained for minor variants complicated data interpretation, an in-depth insight into the charge variants pattern of MB02 compared to Avastin® was obtained, contributing to a better understanding of modifications associated to microheterogeneity. To our knowledge, this is the first comparative analytical study of individual charge variants of a bevacizumab biosimilar following a head-to head approach and the most comprehensive N-glycosylation assessment of IgG1 charge variants. Although modifications related to N- and C-terminal, N-glycans, size heterogeneity or deamidation were specifically enriched among low abundant charge variants, they did not affect binding affinity to VEGF or FcRn and in vitro potency compared with the main species or unfractionated material.

Similarity demonstrated between isolated charge variants of MB02, a biosimilar of bevacizumab, and Avastin® following extended physicochemical and functional characterization.

The majority of recombinant mAb products contain heterogeneous charge variants, commonly the result of post-translational modifications occurring during cell culture and accumulated during production, formulation and storage. MB02 is a biosimilar mAb to bevacizumab. Similarity data of charge variants for biosimilars against its reference products must be generated to demonstrate consistency in product quality and to ensure efficacy and safety. The goal of this work was to isolate seven charge variants of MB02 and Avastin® by semi-preparative cation exchange chromatography followed by purity test and extended analytical characterization to prove similarity. Although poor purity obtained for minor variants complicated data interpretation, an in-depth insight into the charge variants pattern of MB02 compared to Avastin® was obtained, contributing to a better understanding of modifications associated to microheterogeneity. To our knowledge, this is the first comparative analytical study of individual charge variants of a bevacizumab biosimilar following a head-to head approach and the most comprehensive N-glycosylation assessment of IgG1 charge variants. Although modifications related to N- and C-terminal, N-glycans, size heterogeneity or deamidation were specifically enriched among low abundant charge variants, they did not affect binding affinity to VEGF or FcRn and in vitro potency compared with the main species or unfractionated material.

Psoralen and Ultraviolet A Light Treatment Directly Affects Phosphatidylinositol 3-Kinase Signal Transduction by Altering Plasma Membrane Packing.

Psoralen and ultraviolet A light (PUVA) are used to kill pathogens in blood products and as a treatment of aberrant cell proliferation in dermatitis, cutaneous T-cell lymphoma, and graft-versus-host disease. DNA damage is well described, but the direct effects of PUVA on cell signal transduction are poorly understood. Because platelets are anucleate and contain archetypal signal transduction machinery, they are ideally suited to address this. Lipidomics on platelet membrane extracts showed that psoralen forms adducts with unsaturated carbon bonds of fatty acyls in all major phospholipid classes after PUVA. Such adducts increased lipid packing as measured by a blue shift of an environment-sensitive fluorescent probe in model liposomes. Furthermore, the interaction of these liposomes with lipid order-sensitive proteins like amphipathic lipid-packing sensor and α-synuclein was inhibited by PUVA. In platelets, PUVA caused poor membrane binding of Akt and Bruton's tyrosine kinase effectors following activation of the collagen glycoprotein VI and thrombin protease-activated receptor (PAR) 1. This resulted in defective Akt phosphorylation despite unaltered phosphatidylinositol 3,4,5-trisphosphate levels. Downstream integrin activation was furthermore affected similarly by PUVA following PAR1 (effective half-maximal concentration (EC50), 8.4 ± 1.1 versus 4.3 ± 1.1 µm) and glycoprotein VI (EC50, 1.61 ± 0.85 versus 0.26 ± 0.21 µg/ml) but not PAR4 (EC50, 50 ± 1 versus 58 ± 1 µm) signal transduction. Our findings were confirmed in T-cells from graft-versus-host disease patients treated with extracorporeal photopheresis, a form of systemic PUVA. In conclusion, PUVA increases the order of lipid phases by covalent modification of phospholipids, thereby inhibiting membrane recruitment of effector kinases.

Profiling over 1500 lipids in induced lung sputum and the implications in studying lung diseases.

Induced lung sputum is a valuable matrix in the study of respiratory diseases. Although the methodology of sputum collection has evolved to a point where it is repeatable and responsive to inflammation, its use in molecular profiling studies is still limited. Here, an in-depth lipid profiling of induced lung sputum using high-resolution liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-Q-TOF MS) is described. An enormous complexity in lipid composition could be revealed. Over 1500 intact lipids, originating from 6 major lipid classes, have been accurately identified in 120 µL of induced sputum. By number and measured intensity, glycerophospholipids represent the largest lipid class, followed by sphingolipids, glycerolipids, fatty acyls, sterol lipids, and prenol lipids. Several prenol lipids, originating from tobacco, could be detected in the lung sputum of smokers. To illustrate the utility of the methodology in studying respiratory diseases, a comparative lipid screening was performed on lung sputum extracts in order to study the effect of Chronic Obstructive Pulmonary Disease (COPD) on the lung barrier lipidome. Results show that sphingolipid expression in induced sputum significantly differs between smokers with and without COPD.

Comprehensive two-dimensional liquid chromatography of therapeutic monoclonal antibody digests.

Comprehensive two-dimensional liquid chromatography (LC×LC) is here proposed as a novel tool for peptide mapping of therapeutic monoclonal antibodies in both R&D and routine (QA/QC) environments. This is illustrated by the analysis of the tryptic digest of trastuzumab (Herceptin) applying a commercially available two-dimensional 2D-LC system. Three different LC×LC combinations, i.e., strong cation-exchange × reversed-phase (SCX×RP), reversed-phase × reversed-phase (RP×RP), and hydrophilic interaction × reversed-phase (HILIC×RP), are reported. Detection was carried out using both UV detection (DAD) and mass spectrometry (MS). Several challenges related to the application of LC×LC in peptide mapping and the hyphenation to MS are addressed. The applicability of LC×LC in the assessment of identity, purity, and comparability is demonstrated by the analysis of different Herceptin innovator production batches, a Herceptin biosimilar in development and of stressed samples. The described methodology was shown to be precise in terms of peak volume and (2)D retention time opening interesting perspectives for use in QA/QC testing.

Untargeted lipidomic analysis in chronic obstructive pulmonary disease. Uncovering sphingolipids.

RATIONALE: Cigarette smoke is the major risk factor in the development of chronic obstructive pulmonary disease (COPD). Lipidomics is a novel and emerging research field that may provide new insights in the origins of chronic inflammatory diseases, such as COPD. OBJECTIVES: To investigate whether expression of the sputum lipidome is affected by COPD or cigarette smoking. METHODS: Lipid expression was investigated with liquid chromatography and high-resolution quadrupole time-of-flight mass spectrometry in induced sputum comparing smokers with and without COPD, and never-smokers. Changes in lipid expression after 2-month smoking cessation were investigated in smokers with and without COPD. MEASUREMENTS AND MAIN RESULTS: More than 1,500 lipid compounds were identified in sputum. The class of sphingolipids was significantly higher expressed in smokers with COPD than in smokers without COPD. At single compound level, 168 sphingolipids, 36 phosphatidylethanolamine lipids, and 5 tobacco-related compounds were significantly higher expressed in smokers with COPD compared with smokers without COPD. The 13 lipids with a high fold change between smokers with and without COPD showed high correlations with lower lung function and inflammation in sputum. Twenty (glyco)sphingolipids and six tobacco-related compounds were higher expressed in smokers without COPD compared with never-smokers. Two-month smoking cessation reduced expression of 26 sphingolipids in smokers with and without COPD. CONCLUSIONS: Expression of lipids from the sphingolipid pathway is higher in smokers with COPD compared with smokers without COPD. Considering their potential biologic properties, they may play a role in the pathogenesis of COPD.

Evaluation of size-exclusion chromatography, multi-angle light scattering detection and mass photometry for the characterization of mRNA.

The recent approval of messenger ribonucleic acid (mRNA) as vaccine to combat the COVID-19 pandemic has been a scientific turning point. Today, the applicability of mRNA is being demonstrated beyond infectious diseases, for example in cancer immunotherapy, protein replacement therapy and gene editing. mRNA is produced by in vitro transcription (IVT) from a linear DNA template and modified at the 3' and 5' ends to improve translational efficiency and stability. Co-existing impurities such as RNA fragments and double-stranded RNA (dsRNA), amongst others, can drastically impact mRNA quality and efficacy. In this study, size-exclusion chromatography (SEC) is evaluated for the characterization of IVT-mRNA. The effect of mobile phase composition (ionic strength and organic modifier), pH, column temperature and pore size (300 Å, 1000 Å, and 2000 Å) on the separation performance and structural integrity of IVT-mRNA varying in size is described. Non-replicating, self-amplifying (saRNA), temperature degraded, and ribonuclease (RNase) digested mRNA, the latter to characterize the 3' poly(A) tail, were included in the study. Beyond ultraviolet (UV) detection, refractive index (RI) and multi-angle light scattering (MALS) detection were implemented to accurately determine molecular weight (MW) of mRNA. Finally, mass photometry is introduced as a complementary methodology to study mRNA under native conditions.

Multidimensional LC-MS with 1D multi-method option and parallel middle-up and bottom-up MS acquisition for in-depth characterization of antibodies.

Monoclonal antibodies (mAbs) are large and highly heterogeneous species typically characterized using a plethora of analytical methodologies. There is a trend within the biopharmaceutical industry to combine several of these methods in one analytical platform to simultaneously assess multiple structural attributes. Here, a protein analyzer for the fully automated middle-up and bottom-up liquid chromatography-mass spectrometry (LC-MS) analysis of charge, size and hydrophobic variants is described. The multidimensional set-up combines a multi-method option in the first dimension (1D) (choice between size exclusion - SEC, cation exchange - CEX or hydrophobic interaction chromatography - HIC) with second dimension (2D) on-column reversed-phase (RPLC) based desalting, denaturation and reduction prior to middle-up LC-MS analysis of collected 1D peaks and parallel on-column trypsin digestion of denatured and reduced peaks in the third dimension (3D) followed by bottom-up LC-MS analysis in the fourth dimension (4D). The versatile and comprehensive workflow is applied to the characterization of charge, hydrophobic and size heterogeneities associated with an engineered Fc fragment and is complemented with hydrogen-deuterium exchange (HDX) MS and FcRn affinity chromatography - native MS to explain observations in a structural/functional context.

Critical role of aldehydes in cigarette smoke-induced acute airway inflammation.

BACKGROUND: Cigarette smoking (CS) is the most important risk factor for COPD, which is associated with neutrophilic airway inflammation. We hypothesize, that highly reactive aldehydes are critical for CS-induced neutrophilic airway inflammation. METHODS: BALB/c mice were exposed to CS, water filtered CS (WF-CS) or air for 5 days. Levels of total particulate matter (TPM) and aldehydes in CS and WF-CS were measured. Six hours after the last exposure, inflammatory cells and cytokine levels were measured in lung tissue and bronchoalveolar lavage fluid (BALF). Furthermore, Beas-2b bronchial epithelial cells were exposed to CS extract (CSE) or WF-CS extract (WF-CSE) in the absence or presence of the aldehyde acrolein and IL-8 production was measured after 24 hrs. RESULTS: Compared to CS, in WF-CS strongly decreased (CS; 271.1 ± 41.5 µM, WF-CS; 58.5 ± 8.2 µM) levels of aldehydes were present whereas levels of TPM were only slightly reduced (CS; 20.78 ± 0.59 mg, WF-CS; 16.38 ± 0.36 mg). The numbers of mononuclear cells in BALF (p<0.01) and lung tissue (p<0.01) were significantly increased in the CS- and WF-CS-exposed mice compared to air control mice. Interestingly, the numbers of neutrophils (p<0.001) in BALF and neutrophils and eosinophils (p<0.05) in lung tissue were significantly increased in the CS-exposed but not in WF-CS-exposed mice as compared to air control mice. Levels of the neutrophil and eosinophil chemoattractants KC, MCP-1, MIP-1α and IL-5 were all significantly increased in lung tissue from CS-exposed mice compared to both WF-CS-exposed and air control mice. Interestingly, depletion of aldehydes in WF-CS extract significantly reduced IL-8 production in Beas-2b as compared to CSE, which could be restored by the aldehyde acrolein. CONCLUSION: Aldehydes present in CS play a critical role in inflammatory cytokine production and neutrophilic- but not mononuclear airway inflammation.

Characterization of mAb size heterogeneity originating from a cysteine to tyrosine substitution using denaturing and native LC-MS.

Upon assessing the comparability between a biosimilar mAb and its reference product by non-reducing CE-SDS, increased levels of a heavy-heavy-light chain (HHL) variant, present as a low molecular weight (LMW) peak, were observed. RPLC-MS applied at top, middle-up and bottom-up level revealed the existence of Cys-to-Tyr substitutions, predominantly at position HC226 involved in connecting LC and HC, explaining the abundant HHL levels. Antigen binding was not impacted by the presence of this size variant suggesting a non-covalent association of Tyr substituted HHL and LC. The latter complex is not maintained in the denaturing conditions associated with CE-SDS and RPLC-MS. Its existence could, nevertheless, be confirmed by native SEC-MS which preserves non-covalent protein interactions during separation and electrospray ionization. Amino acid analysis furthermore demonstrated a depletion of Cys during the fed-batch process indicating that the observed size/sequence variant is not of genetic but rather of metabolic origin. Native SEC-MS showed that supplementing the cell culture medium with Cys halts misincorporation of Tyr and promotes the formation of the desired mAb structure. To the best of our knowledge, Cys-to-Tyr substitutions preventing interchain disulfide bridge formation have not been described earlier. This observation adds to the impressive structural heterogeneity reported to date for mAbs.

Exploring the possibilities of cryogenic cooling in liquid chromatography for biological applications: a proof of principle.

The possibilities to use cryogenic cooling to trap components in liquid chromatography was investigated. In a first step, van 't Hoff plots were measured with a reversed-phase column using the temperature control unit of a conventional high performance liquid chromatography (HPLC) system to gain insight in the retention behavior of proteins at low temperatures. It was estimated that retention factors in the range of k = 10(4) could be achieved at T = -20 °C for lysozyme, indicating that temperature is a usable parameter to trap components in LC. In a next step, trapping experiments were carried out on a nano-LC system, equipped with a UV-detector, using a commercial reversed-phase column. An in-house built setup, allowing cooling of a segment of the column down to temperatures below T = -20 °C, was used to trap components. Experiments were conducted under isocratic and gradient conditions with methanol as organic solvent. It is demonstrated that, by thermally trapping and elution of components, an enhanced S/N ratio and decreased peak widths can be obtained. At the same time, a significant increase in pressure drop occurs during the cooling process. Limitations and benefits of the technique are further discussed.

Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry.

An LC-MS based method for the profiling and characterization of ceramide species in the upper layer of human skin is described. Ceramide samples, collected by tape stripping of human skin, were analyzed by reversed-phase liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry operated in both positive and negative electrospray ionization mode. All known classes of ceramides could be measured in a repeatable manner. Furthermore, the data set showed several undiscovered ceramides, including a class with four hydroxyl functionalities in its sphingoid base. High-resolution MS/MS fragmentation spectra revealed that each identified ceramide species is composed of several skeletal isomers due to variation in carbon length of the respective sphingoid bases and fatty acyl building blocks. The resulting variety in skeletal isomers has not been previously demonstrated. It is estimated that over 1000 unique ceramide structures could be elucidated in human stratum corneum. Ceramide species with an even and odd number of carbon atoms in both chains were detected in all ceramide classes. Acid hydrolysis of the ceramides, followed by LC-MS analysis of the end-products, confirmed the observed distribution of both sphingoid bases and fatty acyl groups in skin ceramides. The study resulted in an accurate mass retention time library for targeted profiling of skin ceramides. It is furthermore demonstrated that targeted data processing results in an improved repeatability versus untargeted data processing (72.92% versus 62.12% of species display an RSD < 15%).

Divergent synthetic approach to 6''-modified α-GalCer analogues.

A synthetic approach is presented for the synthesis of galacturonic acid and D-fucosyl modified KRN7000. The approach allows for late-stage functionalisation of both the sugar 6''-OH and the sphingosine amino groups, which enables convenient synthesis of promising 6''-modified KRN7000 analogues.

Monoclonal antibody charge variant characterization by fully automated four-dimensional liquid chromatography-mass spectrometry.

Fully automated characterization of monoclonal antibody (mAb) charge variants using four-dimensional liquid chromatography-mass spectrometry (4D-LC-MS) is reported and illustrated. Charge variants resolved by cation-exchange chromatography (CEX) using a salt- or pH-gradient are collected in loops installed on a multiple heart-cutting valve and consequently subjected to online desalting, denaturation, reduction and trypsin digestion prior to LC-MS based peptide mapping. This innovation which substantially reduces turnaround time, sample manipulation, loss and artefacts and increases information gathering, is described in great technical detail, and applied to characterize the charge heterogeneity associated with three therapeutic mAbs. Sequence coverages > 95% are obtained for major and minor charge variants (> 1.0%). Post-translational modifications (PTMs) and modification sites are readily revealed in a repeatable manner including unstable succinimide intermediates which are not maintained when performing classical in-solution overnight digestion of offline collected CEX peaks.

Middle-up characterization of monoclonal antibodies by online reduction liquid chromatography-mass spectrometry.

This study describes the fully automated middle-up characterization of monoclonal antibodies (mAbs) and next-generation variants by online reduction liquid chromatography-mass spectrometry (LC-MS). Proteins were trapped on-column and subjected to online desalting, denaturation and reduction prior to reversed phase elution of the created subunits in the MS. The evaluation of more than 20 different therapeutic proteins including full length mAbs (subclasses IgG1, IgG2 and IgG4), bispecific antibodies, antibody fragments, fusion proteins and antibody-drug conjugates (ADC) revealed that the online reduction method is as powerful as the widely applied offline sample preparation with dithiothreitol (DTT) as reducing agent and guanidine hydrochloride (Gnd.HCl) as denaturant and tackles some major disadvantages associated with the latter method, i.e. corrosion of stainless steel components, adduct formation impacting spectral quality and sample stability. The value of the online reduction LC-MS method is also enforced by its ability to reveal unstable antibody variants such as succinimide intermediates of asparagine deamidation and aspartic acid isomerization which are often lost when using the offline sample preparation method. The performance of the online reduction LC-MS set-up was verified and it was revealed that the method is precise with RSD values below 0.25% and 3.0% for retention time and area, respectively. Carry-over is within acceptable limits (< 0.5%) and the reducing buffer is stable up to 24 hours.

Quantitative N-Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis.

The identification and accurate quantitation of the various glycoforms contained in therapeutic monoclonal antibodies (mAbs) is one of the main analytical needs in the biopharmaceutical industry, and glycosylation represents a crucial critical quality attribute (CQA) that needs to be addressed. Currently, the reference method for performing such identification/quantitation consists of the release of the N-glycan moieties from the mAb, their labelling with a specific dye (e.g., 2-AB or RFMS) and their analysis by HILIC-FLD or HILIC-MS. In this contribution, the potential of a new cost- and time-effective analytical approach performed at the protein subunit level (middle-up) was investigated for quantitative purposes and compared with the reference methods. The robustness of the approach was first demonstrated by performing the relative quantification of the glycoforms related to a well characterized mAb, namely adalimumab. Then, the workflow was applied to various glyco-engineered mAb products (i.e., obinutuzumab, benralizumab and atezolizumab). Finally, the glycosylation pattern of infliximab (Remicade®) was assessed and compared to two of its commercially available biosimilars (Remsima® and Inflectra®). The middle-up analysis proved to provide accurate quantitation results and has the added potential to be used as multi-attribute monitoring method.

Multimodal biomarker discovery for active Onchocerca volvulus infection.

The neglected tropical disease onchocerciasis, or river blindness, is caused by infection with the filarial nematode Onchocerca volvulus. Current estimates indicate that 17 million people are infected worldwide, the majority of them living in Africa. Today there are no non-invasive tests available that can detect ongoing infection, and that can be used for effective monitoring of elimination programs. In addition, to enable pharmacodynamic studies with novel macrofilaricide drug candidates, surrogate endpoints and efficacy biomarkers are needed but are non-existent. We describe the use of a multimodal untargeted mass spectrometry-based approach (metabolomics and lipidomics) to identify onchocerciasis-associated metabolites in urine and plasma, and of specific lipid features in plasma of infected individuals (O. volvulus infected cases: 68 individuals with palpable nodules; lymphatic filariasis cases: 8 individuals; non-endemic controls: 20 individuals). This work resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine (CCG) as biomarker for O. volvulus. During the targeted validation study, metabolite-specific cutoffs were determined (inosine: 34.2 ng/ml; hypoxanthine: 1380 ng/ml; CCG: 29.7 ng/ml) and sensitivity and specificity profiles were established. Subsequent evaluation of these biomarkers in a non-endemic population from a different geographical region invalidated the urine metabolite CCG as biomarker for O. volvulus. The plasma metabolites inosine and hypoxanthine were confirmed as biomarkers for filarial infection. With the availability of targeted LC-MS procedures, the full potential of these 2 biomarkers in macrofilaricide clinical trials, MDA efficacy surveys, and epidemiological transmission studies can be investigated.