Capillary electrophoresis analysis of industrial galactooligosaccharides.

Galactooligosaccharides are added to infant formula to simulate some of the benefits associated with human milk oligosaccharides, in particular to modulate the gut microbiota. During our study the galactooligosaccharide content of an industrial GOS ingredient was determined by differential enzymatic digestion using amyloglucosidase and ß-galactosidase. The resulting digests were fluorophore labeled and analyzed by capillary gel electrophoresis with laser induced fluorescence detection. Quantification of the results were based on a lactose calibration curve. Utilizing this approach, the galactooligosaccharide concentration of the sample was determined as 37.23 g/100 g, very similar to earlier HPLC results, but requiring only 20 min separation time. The CGE-LIF method in conjunction with the differential enzymatic digestion protocol demonstrated in this paper offers a rapid and easy to use method to measure galactooligosaccharides and should be applicable to the determination of GOS in infant formulas and other products.

Analysis of Peptides and Proteins by Native and SDS Capillary Gel Electrophoresis Coupled to Electrospray Ionization Mass Spectrometry via a Closed-Circuit Coaxial Sheath Flow Reactor Interface.

A simple and widely applicable coaxial sheath flow reactor interface (CSFRI) is introduced for easy and robust connection of liquid-phase microseparation methods to mass spectrometric detection, especially for capillary gel electrophoresis analysis of proteins and peptides including SDS-protein complexes. The interface readily accommodated post-column reactions prior to MS detection. It was demonstrated that this novel closed-circuit connection allowed the utilization of non-MS friendly buffer components without significant ion suppression and supported stable electrospray. In SDS capillary agarose gel electrophoresis mode, addition of γ-cyclodextrin to the sheath liquid efficiently removed the SDS content of the sample and the background electrolyte in the flow reactor section by inclusion complexation, while maintaining good separation efficiency and decreasing ion suppression.

Introduction of a Capillary Gel Electrophoresis-Based Workflow for Biotherapeutics Characterization: Size, Charge, and N-Glycosylation Variant Analysis of Bamlanivimab, an Anti-SARS-CoV-2 Product.

Coronavirus Disease 2019 (COVID-19) is a major public health problem worldwide with 5-10% hospitalization and 2-3% global mortality rates at the time of this publication. The disease is caused by a betacoronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The receptor-binding domain (RBD) of the Spike protein expressed on the surface of the virus plays a key role in the viral entry into the host cell via the angiotensin-converting enzyme 2 receptor. Neutralizing monoclonal antibodies having the RBD as a target have the ability to inhibit angiotensin-converting enzyme 2 (ACE2) receptor binding, therefore, prevent SARS-CoV-2 infection, represent a promising pharmacological strategy. Bamlanivimab is the first anti-spike neutralizing monoclonal antibody, which got an emergency use authorization from the FDA for COVID-19 treatment. Albeit, bamlanivimab is primarily a neutralizing mAb, some of its effector function related activity was also emphasized. The effector function of antibody therapeutics is greatly affected by their N-linked carbohydrates at the conserved Fc region, possibly influenced by the manufacturing process. Various capillary gel electrophoresis methods are widely accepted in the biopharmaceutical industry for the characterization of therapeutic antibodies. In this paper we introduce a capillary gel electrophoresis based workflow for 1) size heterogeneity analysis to determine the presence/absence of the non-glycosylated heavy chain (NGHC) fragment (SDS-CGE); 2) capillary gel isoelectric focusing for possible N-glycosylation mediated charge heterogeneity determination, e.g., for excess sialylation and finally, 3) capillary gel electrophoresis for N-glycosylation profiling and sequencing. Our results have shown the presence of negligible amount of non-glycosylated heavy chain (NGHC) while 25% acidic charge variants were detected. Comprehensive N-glycosylation characterization revealed the occurrence of approximately 8.2% core-afucosylated complex and 17% galactosylated N-linked oligosaccharides, suggesting the possible existence of antibody dependent cell mediated cytotoxicity (ADCC) effector function in addition to the generally considered neutralizing effect of this particular therapeutic antibody molecule.

Capillary Sodium Dodecyl Sulfate Agarose Gel Electrophoresis of Proteins.

Capillary sodium dodecyl sulfate gel electrophoresis has long been used for the analysis of proteins, mostly either with entangled polymer networks or translationally cross-linked gels. In this paper capillary agarose gel electrophoresis is introduced for the separation of low molecular weight immunoglobulin subunits. The light (LC~24 kDa) and heavy (HC~50 kDa) chain fragments of a monoclonal antibody therapeutic drug were used to optimize the sieving matrix composition of the agarose/Tris-borate-EDTA (TBE) systems. The agarose and boric acid contents were systematically varied between 0.2-1.0% and 320-640 mM, respectively. The influence of several physical parameters such as viscosity and electroosmotic flow were also investigated, the latter to shed light on its effect on the electrokinetic injection bias. Three dimensional Ferguson plots were utilized to better understand the sieving performance of the various agarose/TBE ratio gels, especially relying on their slope (retardation coefficient, KR) value differences. The best resolution between the LC and non-glycosylated HC IgG subunits was obtained by utilizing the molecular sieving effect of the 1% agarose/320 mM boric acid composition (ΔKR = 0.035). On the other hand, the 0.8% agarose/640 mM boric acid gel showed the highest separation power between the similar molecular weight, but different surface charge density non-glycosylated HC and HC fragments (ΔKR = 0.005). It is important to note that the agarose-based gel-buffer systems did not require any capillary regeneration steps between runs other than simple replenishment of the sieving matrix, significantly speeding up analysis cycle time.

N-glycosylation structure - function characterization of omalizumab, an anti-asthma biotherapeutic product.

Omalizumab, a glycoprotein based biotherapeutics, is one of the most frequently used targeted antibody biopharmaceutical to reduce asthma exacerbations, improve lung function and reduce oral corticosteroid use. The effector function and clearance time of such glycoprotein drugs is affected by their N-glycosylation, that defines the required administration frequency to improve the quality of life in appropriately selected patients. Therefore, the glycosylation of biologics is an important critical quality attribute (CQA). The profile of asparagine linked carbohydrates is greatly dependent on the manufacturing process. Even a small deviation may have a major effect on the structure and therefore the function of the biotherapeutic product. For this reason, comprehensive N-glycosylation analysis is of high importance during production and release. Capillary electrophoresis (CE) is one of the frequently used tools to characterize protein therapeutics and utilized by the biopharmaceutical industry for protein and glycan level analysis, which are key parts both for drug development and quality control. To reveal important structure - function relationships, characterization of omalizumab is presented using capillary SDS gel electrophoresis with UV detection at the protein level and capillary gel electrophoresis with laser induced fluorescent detection at the N-linked carbohydrate level. This latter technique was also used for oligosaccharide sequencing for glycan structure validation. The results suggested no ADCC function - structure relationship due to the mostly core fucosylated biantennary glycans found. However, the presence of the high mannose structures probably affects the clearance rate of the drug.

Ultrafast high-resolution analysis of human milk oligosaccharides by multicapillary gel electrophoresis.

There is recently growing interest towards synthesized human milk oligosaccharides (HMOs) as baby formula additives, and interestingly also as dietary supplements for adults. Currently quite a few manufacturers synthesize HMOs, however, their analysis is challenging, both in resolution and speed. In this paper an ultrafast high-resolution method is introduced for the separation of HMOs by multicapillary gel electrophoresis. Two gel compositions were evaluated with complementary resolving power. One was a conventionally used industrial standard carbohydrate separation matrix, resolving oligosaccharides according to their charge to hydrodynamic volume ratios. The other one was a borate-buffered dextran gel, which utilized the secondary equilibrium of the borate-vicinal diol complexation to enhance resolution. Considering the rapid analysis time and multiplexing (12-channel system), a 96 well sample plate can be analyzed in less than 80 min with the conventional type carbohydrate separation matrix and in less than one hour with the borate-buffered dextran gel. Exploiting the one fluorophore per molecule labeling stoichiometry, the limit of detection (S/N > 3) and limit of quantitation (S/N > 10) were determined as 0.025 and 0.100 mg/mL, respectively, with good linearity. Based on the calibration plot, the quantities of several low concentration HMOs were determined from a human milk sample.

The effect of simulated space radiation on the N-glycosylation of human immunoglobulin G1.

On a roundtrip to Mars, astronauts are expectedly exposed to an approximate amount of radiation that exceeds the lifetime limits on Earth. This elevated radiation dose is mainly due to Galactic Cosmic Rays and Solar Particle Events. Specific patterns of the N-glycosylation of human Igs have already been associated with various ailments such as autoimmune diseases, malignant transformation, chronic inflammation, and ageing. The focus of our work was to investigate the effect of low-energy proton irradiation on the IgG N-glycosylation profile with the goal if disease associated changes could be detected during space travel and not altered by space radiation. Two ionization sources were used during the experiments, a Van de Graaff generator for the irradiation of solidified hIgG samples in vacuum, and a Tandetron accelerator to irradiate hIgG samples in aqueous solution form. Structural carbohydrate analysis was accomplished by CE with laser induced fluorescent detection to determine the effects of simulated space radiation on N-glycosylation of hIgG1 samples. Our results revealed that even several thousand times higher radiation doses that of astronauts can suffer during long duration missions beyond the shielding environment of Low Earth Orbit, no changes were observed in hIgG1 N-glycosylation. Consequently, changes in N-linked carbohydrate profile of IgG1 can be used as molecular diagnostic tools in space.