Current Molecular Medicine - Volume 20, Issue 10, 2020

Background: Immunoglobulin G and A, transferrin, haptoglobin and alpha-1- antitrypsin represent approximately 85% of the human serum glycoproteome and their N-glycosylation analysis may lead to the discovery of important molecular disease markers. However, due to the labile nature of the sialic acid residues, the desialylated subset of the serum N-glycoproteome has been traditionally utilized for diagnostic applications. Objective: Creating a five-protein model to deconstruct the overall N-glycosylation fingerprints in inflammatory and malignant lung diseases. Methods: The N-glycan pool of human serum and the five high abundant serum glycoproteins were analyzed. Simultaneous endoglycosidase/sialidase digestion was followed by fluorophore labeling and separation by CE-LIF to establish the model. Pooled serum samples from patients with COPD, lung cancer (LC) and their comorbidity were all analyzed. Results: Nine significant (>1%) asialo-N-glycan structures were identified both in human serum and the standard protein mixture. The core-fucosylated-agalacto-biantennary glycan differentiated COPD and LC and both from the control and the comorbidity groups. Decrease in the core-fucosylated-agalacto-biantennary-bisecting, monogalacto and bigalacto structures differentiated all disease groups from the control. The significant increase of the fucosylated-galactosylated-triantennary structure was highly specific for LC, to a medium extent for COPD and a lesser extent for comorbidity. Also, some increase in the afucosylated-galactosylated-biantennary structure in all three disease types and afucosylated-galactosylated-triantennary structures in COPD and LC were observed in comparison to the control group. Conclusion: Our results suggested that changes in the desialylated human serum Nglycome hold glycoprotein specific molecular diagnostic potential for malignant and inflammatory lung diseases, which can be modeled with the five-protein mixture.

Background: Type 2 diabetes (T2DM) and colorectal cancer (CRC) are both known to modulate gene expression patterns in peripheral blood leukocytes (PBLs). Objective: As T2DM has been shown to increase the incidence of CRC, we were prompted to check whether diabetes affects mRNA signatures in PBLs isolated from CRC patients. Methods: Twenty-two patients were recruited to the study and classified into four cohorts (healthy controls; T2DM; CRC; CRC and T2DM). Relative expression levels of 573 cell signaling gene transcripts were determined by reverse transcription real-time PCR assays run on low-density OpenArray platforms. Enrichment analysis was performed with the g:GOSt profiling tool to order differentially expressed genes into functional pathways. Results: 49 genes were found to be significantly up- or downregulated in tumorous diabetic individuals as compared to tumor-free diabetic controls, while 11 transcripts were differentially regulated in patients with CRC versus healthy, tumor-free and nondiabetic controls. Importantly, these gene sets were completely distinct, implying that diabetes exerts a profound influence on the transcription of signaling genes in CRC. The top 5 genes showing the most significant expression differences in both contexts were PCK2, MAPK9, CCND1, HMBS, TLR3 (p≤0.0040) and CREBBP, PPIA, NFKBIL1, MDM2 and SELPLG (p≤0.0121), respectively. Functional analysis revealed that most significantly affected pathways were cytokine, interleukin and PI3K/Akt/mTOR signaling cascades as well as mitotic regulation. Conclusion: We propose that differentially expressed genes listed above might be potential biomarkers of CRC and should be studied further on larger patient groups. Diabetes might promote colorectal carcinogenesis by impairing signaling pathways in PBLs.

Aims: Application of capillary electrophoresis with laser induced fluorescence detection (CE-LIF) to identify the N-glycosylation structures of serum and saliva IgA from healthy controls and patients with malignant hematological diseases having cytostatic treatment induced mild oral mucosal lesions. Background: Altered N-glycosylation of body fluid glycoproteins can be an effective indicator of most inflammatory processes. Immunoglobulin A (IgA) is the second highest abundant immunoglobulin and has a major role in the immune-defense against potential pathogen attacks. While IgA is abundant in serum, secretory immunoglobulin A (sIgA) is one of the most prevalent proteins in mucosal surfaces, such as in saliva. Objective: Our aim was to investigate the changes of IgA glycosylation in serum and saliva as a response to an administered cytostatic treatment in patients with malignant hematological disorders. Methods: Capillary electrophoresis with laser induced fluorescent detection (CE-LIF) was used to analyze the N-glycosylation profiles of Z(IgA1) partitioned immunoglobulin A in pooled serum and saliva of 10 control subjects and 8 patients with malignant hematological diseases having cytostatic treatment induced mild oral mucosal lesions. Results: Eight of 31 and four of 38 N-glycans in serum and saliva, respectively, showed significant (p<0.05) differences upon comparison to the control group. Thirteen glycans were present in the saliva but not in the serum, on the other hand, six structures were found in the serum samples not present in the saliva. Conclusion: The developed Z(IgA1) partitioning and the high resolution CE-LIF based glyocoanalytical methods provided an efficient and sensitive workflow to detect and monitor IgA glycosylation alterations in serum and saliva with the scope for widespread molecular medicinal use.

Aim: To demonstrate the capabilities of our new capillary electrophoresis – mass spectrometry method, which facilitates highly accurate relative quantitation of modification site occupancy of antibody-ligand (e.g., antibody-drug) conjugates. Background: Antibody-drug conjugates play important roles in medical discovery for imaging and therapeutic intervention. The localization and stoichiometry of the conjugation can affect the orientation, selectivity, specificity, and strength of molecular interactions, influencing biochemical function. Objective: To demonstrate the option to analyze the localization and stoichiometry of antibody-ligand conjugates by using essentially the same method at all levels including ligand infusion, peptide mapping, as well as reduced and intact protein analysis. Materials and Methods: Capillary electrophoresis coupled with electrospray ionization mass spectrometry was used to analyze the antibody-ligand conjugates. Results: We identified three prevalent ligand conjugation sites with estimated stoichiometries of 73, 14, and 6% and an average ligand-antibody ratio of 1.37, illustrating the capabilities of CE-ESI-MS for rapid and efficient characterization of antibody-drug conjugates. Conclusion: The developed multilevel analytical method offers a comprehensive way to determine the localization and stoichiometry of antibody-drug conjugates for molecular medicinal applications. In addition, a significant advantage of the reported approach is the small, hydrophilic, unmodified peptides well separated from the neutrals, which is not common with other liquid phase separation methods such as LC.

Background: Plasmid DNA has been widely used in vaccination as well as in cell and gene therapy. It exists in multiple isoforms, including supercoiled, nicked or open circular and linear forms. Regulatory agencies recommend having more than 80% of the supercoiled isoform for the bulk release of plasmid products; thus, it should be analyzed accordingly. Methods and Results: The traditional analysis method for plasmid DNA is agarose gel electrophoresis. However, due to time-consuming manual sample loading, visualization, and data analysis, it has limitations in obtaining consistently quantitative results. In this short communication, we introduced a fast, sensitive, and robust plasmid analysis method using capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). CGE-LIF analysis of the supercoiled isoform and its open circular counterpart was completed in 20 minutes with excellent sensitivity by using a common fluorescent DNA binding dye. The advantage of the method was demonstrated by the purity analysis of two large plasmids (7 kb and 10 kb). The fully automated sample loading, separation and data analysis featured enhanced assay repeatability and ease of quantitation over agarose gel electrophoresis. Conclusion: As a worked example, analysis of plasmid samples treated at elevated temperature during an accelerated stability test also demonstrated the applicability of CGE-LIF to monitor plasmid topology and possible degradation.

Adeno-associated virus (AAV) is one of the most promising viral gene delivery vectors with long-term gene expression and disease correction, featuring high efficiency and excellent safety in human clinical trials. During the production of AAV vectors, there are several quality control (QC) parameters that should be rigorously monitored to comply with clinical safety and efficacy. This review gives a short summary of the most frequently used AVV production and purification methods, focusing on the analytical techniques applied to determine the full/empty capsid ratio and the integrity of the encapsidated therapeutic DNA of the products.

Adeno-associated virus (AAV) is one of the most promising gene transfer vector types featuring long-term gene expression and low toxicity. The lack of pathogenicity and the availability of many serotypes augmented the applicability of AAV virions in gene therapy applications. The recombinant AAV capsid includes the therapeutic protein-coding transgene as well as a promoter to initiate translation and a poly A sequence portion for stabilization. Current AAV manufacturing technologies, however, cannot guarantee the generation of only full capsids, i.e., including the entire required genome. Partially filled and empty capsids are also part of the product, decreasing in this way the efficacy and safety upon clinical translation. Therefore, rapid, accurate and QC friendly analysis of the full and empty capsid ratio is of high importance during AAV vector manufacturing and release testing. In this paper, an automated capillary isoelectric focusing technique is introduced, readily applicable in the biopharmaceutical industry for fast and efficient determination of the full and empty capsid ratio. The method also reveals information about the proportion of partially filled capsids. For higher resolution (<0.1 pI unit), mixtures of wide and narrow range ampholytes were utilized. The isoelectric point and peak area percentage reproducibility (RSD) of the mixed ampholyte assay were as low as 1.67% and 2.45 % , respectively, requiring only 65 nL of sample volume per injection.

Chondroitin sulfate (CS)-glycosaminoglycans (GAGs) are linear, negatively charged polysaccharides attached to CS proteoglycans that make up a major component of biological matrices throughout both central and peripheral tissues. The position of their attached sulfate groups to the CS disaccharide is predicted to influence protein-glycan interactions and biological function. Although traditional immunohistochemical analysis of CS-GAGs in biological tissues has provided information regarding changes in GAG abundance during developmental and disease states, quantitative analysis of their specific sulfation patterns is limited due to the inherent complexity of separating CS isomers. While methods have been developed to analyze and quantify sulfation isomers using liquid phase separation, new techniques are still needed to elucidate the full biology of CS-GAGs. Here, we examine ion mobility spectrometry and gas-phase hydrogen-deuterium exchange to resolve positional sulfation isomers in the most common sulfated 4S- and 6S-CS disaccharides. The mobilities for these two isomers are highly similar and could not be resolved effectively with any drift gas tested. In contrast, gas-phase hydrogen-deuterium exchange showed very different rates of deuterium uptake with several deuterium exchange reagents, thereby presenting a promising novel and rapid approach for resolving CS isomers.

The market segment of new biological drugs (monoclonal antibodies, fusion proteins, antibody-drug conjugates, and new modality protein therapeutics) is rapidly growing, especially after the patent expiration of the original biologics, initiating the emergence of biosimilars. N-glycosylation of therapeutic proteins has high importance on their stability, safety, immunogenicity, efficacy, and serum half-life. Therefore, Nglycosylation is considered to be one of the critical quality attributes. Consequently, it should be rigorously monitored during the development, manufacturing, and release of glycoprotein biologicals. In this review, first, the regulatory considerations for biosimilars are shortly summarized, followed by conferring the analytical techniques needed for monitoring and characterization of the N-glycosylation of biological drugs. Particular respect is paid to liquid phase separation techniques with high sensitivity and highresolution detection methods, including laser-induced fluorescence and mass spectrometry.

The utilization of N-glycan profiling recently gained high importance in fundamental biomedical and applied clinical research. However, for the time being, no glycan biomarker has been approved for clinical diagnosis by the regulatory agencies due to the lack of verifications on large patient cohorts and suitable analytical technologies. In this paper, the effect of human blood sample handling was studied prior to N-glycosylation profiling by capillary electrophoresis, coupled with high sensitivity fluorescence detection. Special attention was paid to the preservation of sialylated structures because of their important clinical - biological relevance. Our results suggested that it is adequate to refrigerate and store the collected total blood samples prior to analysis to obtain unbiased results. Furthermore, we report on the good practice of serum sample handling in order to prevent decomposition of the sialylated structures. Our findings may promote procedure standardization and easier clinical translation of diagnostic N-glycosylation profiling in molecular medicinal applications.