Purified Pyropia yezoensis Pigment Extract-Based Tandem Dye Synthesis.

Red phycoerythrin (R-PE) is a highly valuable protein found in an edible seaweed, Pyropia yezoensis. It is used extensively in biotechnological applications due to its strong fluorescence and stability in diverse environments. However, the current methods for extracting and purifying R-PE are costly and unsustainable. The aim of the present study was to enhance the financial viability of the process by improving the extraction and purification of R-PE from dried P. yezoensis and to further enhance R-PE value by incorporating it into a tandem dye for molecular biology applications. A combination of ultrafiltration, ion exchange chromatography, and gel filtration yielded concentrated (1 mg·mL-1) R-PE at 99% purity. Using purified PE and Cyanine5 (Cy5), an organic tandem dye, phycoerythrin-Cy5 (PE-Cy5), was subsequently established. In comparison to a commercially available tandem dye, PE-Cy5 exhibited 202.3% stronger fluorescence, rendering it suitable for imaging and analyzes that require high sensitivity, enhanced signal-to-noise ratio, broad dynamic range, or shorter exposure times to minimize potential damage to samples. The techno-economic analysis confirmed the financial feasibility of the innovative technique for the extraction and purification of R-PE and PE-Cy5 production.

Intact quantitation of cysteine-conjugated antibody-drug conjugates using native mass spectrometry.

RATIONALE: A common strategy for antibody-drug conjugate (ADC) quantitation from in vivo study samples involves measurement of total antibody, conjugated ADC, and free payload concentrations using multiple reaction monitoring (MRM) mass spectrometry. This not only provides a limited picture of biotransformation but can also involve lengthy method development. Quantitation of ADCs directly at the intact protein level in native conditions using high-resolution mass spectrometers presents the advantage of measuring exposure readout as well as monitoring the change in average drug-to-antibody ratio (DAR) and in vivo stability of new linker payloads with minimal method development. Furthermore, site-specific cysteine-conjugated ADCs often rely on non-covalent association to retain their quaternary structure, which highlights the unique capabilities of native mass spectrometry (nMS) for intact ADC quantitation. METHODS: We developed an intact quantitation workflow involving three stages: automated affinity purification, nMS analysis, and data processing in batch fashion. The sample preparation method was modified to include only volatile ion-pairing reagents in the buffer systems. A capillary size-exclusion chromatography (SEC) column was coupled to a quadrupole time-of-flight high-resolution mass spectrometer for high-throughput nMS analysis. Samples from two mouse pharmacokinetic (PK) studies were analyzed using both intact quantitation workflow and the conventional MRM-based approach. RESULTS: A linear dynamic range of 5-100 µg/mL was achieved using 20 µL of serum sample volume. The results of mouse in vivo PK measurement using the intact quantitation workflow and the MRM-based approach were compared, revealing excellent method agreement. CONCLUSIONS: We demonstrated the feasibility of utilizing nMS for the quantitation of ADCs at the intact protein level in preclinical PK studies. Our results indicate that this intact quantitation workflow can serve as an alternative generic method for high-throughput analysis, enabling an in-depth understanding of ADC stability and safety in vivo.

Recent research on material-based methods for isolation of extracellular vesicles.

Extracellular vesicles (EVs) are nanoparticles secreted by cells with a closed phospholipid bilayer structure, which can participate in various physiological and pathological processes and have significant clinical value in disease diagnosis, targeted therapy and prognosis assessment. EV isolation methods currently include differential ultracentrifugation, ultrafiltration, size exclusion chromatography, immunoaffinity, polymer co-precipitation and microfluidics. In addition, material-based biochemical or biophysical approaches relying on intrinsic properties of the material or its surface-modified functionalized monomers, demonstrated unique advantages in the efficient isolation of EVs. In order to provide new ideas for the subsequent development of material-based EV isolation methods, this review will focus on the principle, research status and application prospects of material-based EV isolation methods based on different material carriers and functional monomers.

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.

Not All Arms of IgM Are Equal: Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS.

Immunoglobulins M (IgM) are key natural antibodies produced initially in humoral immune response. Due to their large molecular weights and extensive glycosylation loads, IgMs represent a challenging target for conventional mass analysis. Charge detection mass spectrometry (CDMS) may provide a unique approach to tackle heterogeneous IgM assemblies, although this technique can be quite laborious and technically challenging. Here, we describe the use of online size exclusion chromatography (SEC) to automate buffer exchange and sample introduction, and demonstrate its adaptability with Orbitrap-based CDMS. We discuss optimal experimental parameters for online SEC-CDMS experiments, including ion activation, choice of column, and resolution. Using this approach, CDMS histograms containing hundreds of individual ion signals can be obtained in as little as 5 min from single injections of <1 µg of sample. To demonstrate the unique utility of online SEC-CDMS, we performed real-time kinetic monitoring of pentameric IgM digestion by the protease IgMBRAZOR, which cleaves specifically in the hinge region of IgM. Several digestion intermediates corresponding to processive losses of F(ab')2 subunits could be mass-resolved and identified by SEC-CDMS. Interestingly, we find that for the J-chain linked IgM pentamer, cleavage of one of the F(ab')2 subunits is much slower than the other four F(ab')2 subunits, which we attribute to the symmetry-breaking interactions of the J-chain within the pentameric IgM structure. The online SEC-CDMS methodologies described here open new avenues into the higher throughput automated analysis of heterogeneous, high-mass protein assemblies by CDMS.

Using molecular fine structure to identify optimal methods of extracting fungal glycogen.

Glycogen is a highly branched glucose polymer that is an energy storage material in fungi and animals. Extraction of glycogen from its source in a way that minimizes its molecular degradation is essential to investigate its native structure. In this study, the following extraction methods were compared: sucrose gradient density ultracentrifugation, thermal alkali, hot alcohol and hot water extractions. Molecular-size and chain-length distributions of glycogen were measured by size-exclusion chromatography and fluorophore-assisted carbohydrate electrophoresis, respectively. These two fine-structure features are the most likely structural characteristics to be degraded during extraction. The results show that the thermal alkali, hot alcohol and hot water extractions degrade glycogen molecular size and/or chain-length distributions, and that sucrose gradient density ultracentrifugation with neither high temperature nor alkaline treatment is the most suitable method for fungal glycogen extraction.

Glycogen ß-particles surface characterized by a combination of size exclusion chromatography and pyrene excimer fluorescence before and after ß-amylolysis.

Size exclusion chromatography (SEC) and pyrene excimer formation (PEF) experiments were conducted to characterize the local density profile inside a glycogen sample before (Glycogen) and after (Gly-ß-LD) treatment with ß-amylase. These experiments were conducted to assess whether the density at the periphery of the glycogen particles was very high to limit access to proteins involved in the metabolism of glycogen as predicted by the Tier model or low as suggested by the Gilbert model. SEC analysis indicated that the density inside the Glycogen and Gly-ß-LD samples remained constant with particle size and was not affected by ß-amylolysis. Analysis of the PEF experiments conducted on the Glycogen and Gly-ß-LD samples labeled with 1-pyrenebutyric acid showed that the particles have a dense interior and loose corona. The conclusions reached by the SEC and PEF experiments agree with the Gilbert model and have implications for the association of glycogen ß-particles into larger α-particles.

Analysis of aggregation profile of glucagon using SEC-HPLC and FFF-MALS methods.

Recently, the first generic glucagon for injection was approved for the treatment of severe hypoglycemia. Unlike its brand name recombinant glucagon, the generic glucagon is synthetic. Since glucagon has a high propensity to form aggregates in solution, it is essential to assess the aggregation profile of the synthetic glucagon compared to the recombinant glucagon. In this study, two robust separation methods, size-exclusion chromatography (SEC-HPLC) and field-flow fractionation coupled with a multi-angle light scattering detector (FFF-MALS), were employed to characterize generic and brand glucagon aggregation in six lots (three newly released, three expired). The presence of aggregation in samples was determined from the generated chromatograms and analyzed. The study showed that both products have comparable aggregation profiles. The SEC-HPLC demonstrated that in both glucagon versions, the expired lots had a higher percentage of dimers than the newly released lots, but even at expiration, the amount was negligible (∼0.1%). The FFF-MALS method did not detect any dimers or higher molecular weight aggregates. Further evaluation of the detection limit found that FFF-MALS was unable to detect aggregates at amounts lower than 0.5% of total glucagon. The negligible amounts of dimer detected in the generic and brand glucagon indicate that both versions are physically stable and are not prone to aggregation under clinically relevant conditions.

Size exclusion chromatography of biopharmaceutical products: From current practices for proteins to emerging trends for viral vectors, nucleic acids and lipid nanoparticles.

The 21st century has been particularly productive for the biopharmaceutical industry, with the introduction of several classes of innovative therapeutics, such as monoclonal antibodies and related compounds, gene therapy products, and RNA-based modalities. All these new molecules are susceptible to aggregation and fragmentation, which necessitates a size variant analysis for their comprehensive characterization. Size exclusion chromatography (SEC) is one of the reference techniques that can be applied. The analytical techniques for mAbs are now well established and some of them are now emerging for the newer modalities. In this context, the objective of this review article is: i) to provide a short historical background on SEC, ii) to suggest some clear guidelines on the selection of packing material and mobile phase for successful method development in modern SEC; and iii) to highlight recent advances in SEC, such as the use of narrow-bore and micro-bore columns, ultra-wide pore columns, and low-adsorption column hardware. Some important innovations, such as recycling SEC, the coupling of SEC with mass spectrometry, and the use of alternative detectors such as charge detection mass spectrometry and mass photometry are also described. In addition, this review discusses the use of SEC in multidimensional setups and shows some of the most recent advances at the preparative scale. In the third part of the article, the possibility of SEC for the characterization of new modalities is also reviewed. The final objective of this review is to provide a clear summary of opportunities and limitations of SEC for the analysis of different biopharmaceutical products.

Determination of Molecular Dimensions of Carbohydrate Polymers (Polysaccharides) by Size Exclusion Chromatography (SEC).

Calibrated size exclusion chromatography (SEC) is a useful tool for the analysis of molecular dimensions of polysaccharides. The calibration takes place with a set of narrow distributed dextran standards and peak position technique. Adapted columns systems and dissolving processes enable for the adequate separation of carbohydrate polymers. Plant-extracted fructan (a homopolymer with low molar mass and excellent water solubility) and mucilage (differently structured, high molar mass heteropolysaccarides that include existing supramolecular structures, and require a long dissolving time) are presented as examples of the versatility of this technique. Since narrow standards similar to the samples (chemically and structurally) are often unavailable, it must be noted that the obtained molar mass values and distributions by this method are only apparent (relative) values, expressed as dextran equivalents.

Beyond the Surface: A Methodological Exploration of Enzyme Impact along the Cellulose Fiber Cross-Section.

Despite the wide range of analytical tools available for the characterization of cellulose, the in-depth characterization of inhomogeneous, layered cellulose fiber structures remains a challenge. When treating fibers or spinning man-made fibers, the question always arises as to whether the changes in the fiber structure affect only the surface or the entire fiber. Here, we developed an analysis tool based on the sequential limited dissolution of cellulose fiber layers. The method can reveal potential differences in fiber properties along the cross-sectional profile of natural or man-made cellulose fibers. In this analytical approach, carbonyl groups are labeled with a carbonyl selective fluorescence label (CCOA), after which thin fiber layers are sequentially dissolved with the solvent system DMAc/LiCl (9% w/v) and analyzed with size exclusion chromatography coupled with light scattering and fluorescence detection. The analysis of these fractions allowed for the recording of the changes in the chemical structure across the layers, resulting in a detailed cross-sectional profile of the different functionalities and molecular weight distributions. The method was optimized and tested in practice with LPMO (lytic polysaccharide monooxygenase)-treated cotton fibers, where it revealed the depth of fiber modification by the enzyme.

Development of a high throughput oxidation profiling strategy for monoclonal antibody products.

Oxidation is one of the most common degradation pathways of biopharmaceutics, potentially leading to altered product stability, pharmacokinetics, reduced biological activity and/or an increased immunogenicity. However, it is often insufficiently assessed in early development stages, leaving potential molecule liabilities undiscovered. Aim of the present work was the development of a high throughput oxidation profiling strategy, applicable throughout various stages of biopharmaceutical development. The study demonstrates that the combination of multiple stress assays, including peroxide-based, visible light, and metal-catalyzed oxidation (MCO), enables a comprehensive understanding of a mAb's oxidation susceptibility. The most effective parameters to evaluate oxidation in a high-throughput screening workflow are aggregation, tryptophan oxidation and changes in the hydrophobicity profile of the Fc and Fab subunit measured via Size Exclusion Chromatography, Intrinsic Tryptophan Fluorescence Emission spectroscopy and Reversed-Phase Chromatography subunit analysis, respectively. This oxidation profiling approach is valuable tool to systematically characterize the oxidation susceptibility under relevant conditions, time effective and with minimal sample consumption.

Studying the stability of polymer nanoparticles by size exclusion chromatography of radioactive polymers.

The properties of nanomedicines will influence how they can deliver drugs to patients reproducibly and effectively. For conventional pharmaceutical products, Chemistry, Manufacturing and Control (CMC) documents require monitoring stability and storage conditions. For nanomedicines, studying these important considerations is hindered by a lack of appropriate methods. In this paper, we show how combining radiolabelling with size exclusion chromatography, using a method called SERP (for Size Exclusion of Radioactive Polymers), can inform on the in vitro degradation of polymer nanoparticles. Using nanoparticles composed of biodegradable poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA), we show that SERP is more sensitive than dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) to detect degradation. We also demonstrate that the properties of the polymer composition and the nature of the aqueous buffer affect nanoparticle degradation. Importantly, we show that minute changes in stability that cannot be detected by DLS and NTA impact the pharmacokinetic of nanoparticles injected in vivo. We believe that SERP might prove a valuable method to document and understand the pharmaceutical quality of polymer nanoparticles.

Size-Exclusion Chromatography Combined with Ultrafiltration Efficiently Isolates Extracellular Vesicles from Human Blood Samples in Health and Disease.

There is still a need for an efficient method for the isolation of extracellular vesicles (EVs) from human blood that provides a reliable yield with acceptable purity. Blood is a source of circulating EVs, but soluble proteins and lipoproteins hamper their concentration, isolation, and detection. This study aims to investigate the efficiency of EV isolation and characterization methods not defined as "gold standard". EVs were isolated from human platelet-free plasma (PFP) of patients and healthy donors through size-exclusion chromatography (SEC) combined with ultrafiltration (UF). Then, EVs were characterized using transmission electron microscopy (TEM), imaging flow cytometry (IFC), and nanoparticle tracking analysis (NTA). TEM images showed intact and roundish nanoparticles in pure samples. IFC analysis detected a prevalence of CD63+ EVs compared to CD9+, CD81+, and CD11c+ EVs. NTA confirmed the presence of small EVs with a concentration of ~1010 EVs/mL that were comparable when stratifying the subjects by baseline demographics; conversely, concentration differed according to the health status across healthy donors and patients affected with autoimmune diseases (130 subjects in total, with 65 healthy donors and 65 idiopathic inflammatory myopathy (IIM) patients). Altogether, our data show that a combined EV isolation method, i.e., SEC followed by UF, is a reliable approach to isolate intact EVs with a significant yield from complex fluids, which might characterize disease conditions early.

Effect of Lyophilization on Stability of PEG-Protein Conjugate: A Case Study with Peginterferon alfa-2b

Abstract The purpose of the present study was to develop stable lyophilized formulation of peginterferon alfa-2b which is acquiescent to the short lyophilization process. The present study evaluates the effect of buffering components and cryoprotectant(s) on depegylation of the peginterferon alfa-2b in combination with lyophilization process. Finally, a short lyophilization process was identified which can produce a stable pharmaceutical form of peginterferon alfa-2b without any depegylation during long-term storage. Formulations were analyzed mainly for depegylation by HP-size exclusion chromatography and in-vitro antiviral activity. Residual moisture content in the lyophilized product was also used as a key indicating parameter to check its role with respect to depegylation upon storage under various temperature conditions. It was observed that the peginterferon alfa-2b when formulated in presence of cryoprotectant like sucrose requires longer lyophilization process of about 5 days, irrespective of the buffering components used, to reduce the level of residual moisture content and thereby to produce the stable formulation without depegylation. A stable formulation in presence of high concentration of lactose as a cryoprotectant was developed which can withstand stresses exerted to protein-polymer conjugate during lyophilization phases without any significant depegylation. A short lyophilization process of about 48 hours can be utilized for peginterferon alfa-2b when formulated in presence of lactose as a cryoprotectant through which a stable lyophilized formulation can be produced as against longer process required when sucrose is used a cryoprotectant, which is essential from commercial point of view as lyophilization is a costly process.

Caracterización del polirribosilribitol fosfato por cromatografía de exclusión molecular de alta eficacia con detección ultravioleta / Characterization of polyribosylribitol phosphate by high efficiency size exclusion chromatography with ultraviolet detection

Haemophilus influenzae tipo b es un importante patógeno del hombre causante de varias de las enfermedades invasivas en niños menores de cinco años, contra el cual fueron autorizadas las vacunas glicoconjugadas a partir del polirribosilribitol fosfato. Quimi-Hib® es la primera y única vacuna contra este patógeno que utiliza el polisacárido obtenido por síntesis química. El Ingrediente Farmacéutico Activo es producido por el Centro de Ingeniería Genética y Biotecnología y se obtiene a partir de su conjugación al toxoide tetánico. En el presente reporte se hizo una caracterización del polirribosilribitol fosfato mediante la técnica de cromatografía de exclusión molecular de alta eficacia con detección ultravioleta a 215 nm. En el estudio se evaluaron tres lotes y se determinó el perfil de elución en una columna SuperdexTM 75 10/300 GL Increase con un porciento de pureza de 77,42 ± 8,97 y una masa molar promedio de 7.381 Da ± 210,93. La principal impureza presente en el polirribosilribitol fosfato es el dimetilsulfóxido, disolvente utilizado en la reacción de activación con el éster N-hidroxisuccinimidilo del ácido β-maleimidopropiónico. El polirribosilribitol fosfato se purificó por filtración con un Amicon Ultra-15 de 2.000 Da hasta una pureza de 99,1 por ciento y se conjugó al toxoide tetánico. El rendimiento de la reacción de conjugación con el polisacárido purificado fue de 30,0 por ciento 1,77 el cual no muestra diferencias significativas con el control que fue 33,7 por ciento ± 3,57 demostrándose que el dimetilsulfóxido no afecta el desempeño de la reacción de conjugación(AU)

Haemophilus influenzae type b is an important human pathogen causing some invasive diseases in children less than five years of age. Glycoconjugate vaccines based on polyribosylribitol phosphate have been licensed against this bacterium. Quimi-Hib® is the first and only vaccine against this pathogen using the chemically synthesized polysaccharide. The Active Pharmaceutical Ingredient is produced by the Center for Genetic Engineering and Biotechnology and is obtained from its conjugation to tetanus toxoid. In the present report a characterization of polyribosylribitol phosphate was performed by high performance molecular exclusion chromatography with ultraviolet detection at 215 nm. Three batches were evaluated in the study and the elution profile was determined on a SuperdexTM 75 10/300 GL Increase column with a purity percentage of 77.42 ± 8.97 and an average molecular weight of 7,381 Da ± 210.93. The main impurity present in polyribosylribitol phosphate was dimethylsulfoxide, the solvent used in the activation reaction with N-hydroxysuccinimidyl ester of β-maleimidopropionic acid. Polyribosylribitol phosphate was purified by filtration using a 2,000 Da cut-off Amicon Ultra-15 to a purity of 99.1 percent and conjugated to tetanus toxoid. The yield of the conjugation reaction with the purified polysaccharide was 30.0 percent ± 1.77 which shows no significant difference with the control which was 33.7 percent ± 3.57 demonstrating that dimethylsulfoxide does not affect the performance of the conjugation reaction(AU)

Inline-tandem purification of viruses from cell lysate by agarose-based chromatography.

An efficient chromatography-based virus purification method has been developed and validated for the non-pathogenic infectious virus PRD1. Compared to the conventional method that consists of relatively time-consuming and labour-intensive precipitation and density gradient ultracentrifugation steps, the method developed here is performed in a single flow using tandem-coupled anion exchange and size exclusion chromatography (AIEX-SEC) columns. This inline approach helps to minimize the loss of virus in the process and streamlines time consumption, since no physical transfer of the sample is required between purification steps. In the development process, sample feed composition, dynamic binding capacity and elution conditions for the AIEX resin as well as different exclusion limits for SEC resins were optimized to achieve maximal yield of pure infectious viruses. Utilizing this new approach, a high-quality virus sample was produced from a lysate feed in 320 min with a total yield of 13 mg purified particles per litre of cell lysate, constituting a 3.5-fold yield increase as compared to the conventional method, without compromising the high specific infectivity of the product (6 × 1012 to 7 × 1012 pfu/mg of protein). The yield of infectious viruses of the lysate feed was 54%. The easy scalability of chromatography-based methods provide a direct route to industrial usage without any significant changes needed to be made to the purification regime. This is especially interesting as the method has high potential to be used for purification of various viruses and nanoparticles, including adenovirus.

Understanding the structure and composition of recalcitrant oligosaccharides in hydrolysate using high-throughput biotin-based glycome profiling and mass spectrometry.

Novel Immunological and Mass Spectrometry Methods for Comprehensive Analysis of Recalcitrant Oligosaccharides in AFEX Pretreated Corn Stover. Lignocellulosic biomass is a sustainable alternative to fossil fuel and is extensively used for developing bio-based technologies to produce products such as food, feed, fuel, and chemicals. The key to these technologies is to develop cost competitive processes to convert complex carbohydrates present in plant cell wall to simple sugars such as glucose, xylose, and arabinose. Since lignocellulosic biomass is highly recalcitrant, it must undergo a combination of thermochemical treatment such as Ammonia Fiber Expansion (AFEX), dilute acid (DA), Ionic Liquid (IL) and biological treatment such as enzyme hydrolysis and microbial fermentation to produce desired products. However, when using commercial fungal enzymes during hydrolysis, only 75-85% of the soluble sugars generated are monomeric sugars, while the remaining 15-25% are soluble recalcitrant oligosaccharides that cannot be easily utilized by microorganisms. Previously, we successfully separated and purified the soluble recalcitrant oligosaccharides using a combination of charcoal and celite-based separation followed by size exclusion chromatography and studies their inhibitory properties on enzymes. We discovered that the oligosaccharides with higher degree of polymerization (DP) containing methylated uronic acid substitutions were more recalcitrant towards commercial enzyme mixtures than lower DP and neutral oligosaccharides. Here, we report the use of several complementary techniques that include glycome profiling using plant biomass glycan specific monoclonal antibodies (mAbs) to characterize sugar linkages in plant cell walls and enzymatic hydrolysate, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using structurally-informative diagnostic peaks offered by negative ion post-secondary decay spectra, gas chromatography followed by mass spectrometry (GC-MS) to characterize oligosaccharide sugar linkages with and without derivatization. Since oligosaccharides (DP 4-20) are small, it is challenging to mobilize these molecules for mAbs binding and characterization. To overcome this problem, we have applied a new biotin-coupling based oligosaccharide immobilization method that successfully tagged most of the low DP soluble oligosaccharides on to a micro-plate surface followed by specific linkage analysis using mAbs in a high-throughput system. This new approach will help develop more advanced versions of future high throughput glycome profiling methods that can be used to separate and characterize oligosaccharides present in biomarkers for diagnostic applications.

Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase Proteoforms.

Xylanases are of great value in various industries, including paper, food, and biorefinery. Due to their biotechnological production, these enzymes can contain a variety of post-translational modifications, which may have a profound effect on protein function. Understanding the structure-function relationship can guide the development of products with optimal performance. We have developed a workflow for the structural and functional characterization of an endo-1,4-ß-xylanase (ENDO-I) produced by Aspergillus niger with and without applying thermal stress. This workflow relies on orthogonal native separation techniques to resolve proteoforms. Mass spectrometry and activity assays of separated proteoforms permitted the establishment of structure-function relationships. The separation conditions were focus on balancing efficient separation and protein functionality. We employed size exclusion chromatography (SEC) to separate ENDO-I from other co-expressed proteins. Charge variants were investigated with ion exchange chromatography (IEX) and revealed the presence of low abundant glycated variants in the temperature-stressed material. To obtain better insights into the effect on glycation on function, we enriched for these species using boronate affinity chromatography (BAC). The activity measurements showed lower activity of glycated species compared to the non-modified enzyme. Altogether, this workflow allowed in-depth structural and functional characterization of ENDO-I proteoforms.

Stability assessment of FDA-approved ramucirumab monoclonal antibody; validated SE-HPLC method for degradation pattern evaluation.

Ramucirumab (RAMU) is a recently US Food and Drug Administration-approved monoclonal antibody that is included in various anticancer protocols. It has a structural complexity and high degradation risk that have a significant effect on its safety and effectiveness. The major aim of this work was to assess the degradation pattern of RAMU based on physicochemical characterization. Mechanical agitation, repeated freeze-thaw cycles, pH and temperature were the selected stress conditions to which RAMU samples were subjected. The SE-HPLC method was applied and validated to monitor the RAMU monomer along with its aggregates and/or fragments. The purity of the separated peaks together with system suitability parameters were determined through the calculation of percentage purity and percentage drop in RAMU concentration. The results were interpreted by correlating them with those of dynamic light scattering and reducing and non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Samples incubated at pH 2.0-10.0 and 37°C for up to 4 weeks were analysed, recording detection of reversed phase (RP) aggregates and low molecular weight peptide fragments. Similarly, samples under short-term storage conditions of 4 weeks at different temperatures (-20, 2-8, 25, 37 and 50°C) showed low molecular weight peptide fragments but to a lesser extent. These results highlight the alarming effect on RAMU multidose vial efficacy and safety.