Development of a novel, label-free N-glycan method using charged aerosol detection.

Monoclonal antibodies (mAbs) are complex glycoproteins that are developed for treatment of various therapeutic indications such as cancer and autoimmune diseases. MAbs are glycosylated at conserved asparagine residues (N-X-S/T) of the Fc region at amino acid position 297 of the heavy chain. Glycans are important in governing the functions of efficacy and serum half-life of protein therapeutics and are part of the critical quality attribute panel for release testing. Traditionally, N-linked glycans are released from glycoproteins after denaturation and enzymatic digestion with PNGase F, followed by fluorescent labeling of the liberated glycans. The labeled glycans are then separated using hydrophilic liquid chromatography (HILIC) with fluorescence detection to generate chromatographic profile. Despite decades of use, this strenuous process remains unchanged, utilizing toxic reagents and extended sample preparation time. As an intervention, this report showcases a novel, label-free approach to detect and quantify N-glycans without using fluorescent labeling. Separation of glycans using mixed-mode PGC column along with detection of non-derivatized glycans using charged aerosol detector, the overall turnaround time can be greatly reduced with significant cost savings. The label-free method provides similar quantitative results as the conventional fluorescent labeled method, confirming the validity of the method for product release.

Enhancement of covalent aggregate quantification of protein therapeutics by non-reducing capillary gel electrophoresis using sodium hexadecyl sulfate (CE-SHS).

Capillary gel electrophoresis (CGE) using sodium dodecyl sulfate (CGE-SDS or CE-SDS) is commonly used in the biotechnology industry to assess the purity of a complex therapeutic during manufacturing process optimization and also for commercial release and stability testing. However, for therapeutic proteins mAb-1 and mAb-2, non-reducing (NR) CE-SDS yielded higher than expected % aggregate which considerably lowered its apparent purity relative to the purity reported by other complementary methods, such as Size Exclusion Chromatography (SEC). Furthermore, a strong protein load dependence on aggregate levels was observed which prevented any reasonable assessment of the true purity value. The solution was to supplement SDS with the relatively hydrophobic detergent sodium hexadecyl sulfate (SHS) in the sieving gel buffer matrix which virtually eliminated the protein load-dependence and reduced the % aggregate value to expected levels when compared to SEC. Analytical Ultracentrifugation (AUC) was used to help confirm the accuracy of the SEC results. This work underscored how using detergents other than SDS in CGE applications can be valuable in the commercial biologics space and provided an example of how SEC can be used to confirm the accuracy of CGE data.

Minimization of artifact protein aggregation using tetradecyl sulfate and hexadecyl sulfate in capillary gel electrophoresis under reducing conditions.

In the biopharmaceutical industry, CE-SDS assesses the purity, heterogeneity, and stability of therapeutic proteins. However, for mAb-1 and mAb-2, typical CE-SDS under reducing conditions produced atypical protein peak profiles, which led to biased purity results, thus were not acceptable for biologics manufacturing. This bias was caused by the formation of method-induced higher molecular weight artifacts, the levels of which correlated with protein concentration. Here we show that adding sodium tetradecyl and hexadecyl sulfates to the sample and the sieving gel buffer solutions was required to prevent formation of aggregate artifacts and to maintain detergent:protein uniformity, suggesting their importance during the sample preparation steps of heat denaturation and subsequent cooling as well as during capillary migration. For these proteins, we show that this uniformity was likely due to the ability of these detergents to bind proteins with markedly higher affinities compared to SDS. "CE-SCX S" methods (where CE-SCX S is CGE using detergent composed of a sodium sulfate head group and a hydrocarbon tail, with "CX " representing various tail lengths), were developed with a sodium tetradecyl sulfate sample buffer and a sodium hexadecyl sulfate containing sieving gel buffer that minimized artifacts and provided robust characterization and release results for mAb-1 and mAb-2.

Distinct and combinatorial functions of Jmjd2b/Kdm4b and Jmjd2c/Kdm4c in mouse embryonic stem cell identity.

Self-renewal and pluripotency of embryonic stem cells (ESCs) are established by multiple regulatory pathways operating at several levels. The roles of histone demethylases (HDMs) in these programs are incompletely defined. We conducted a functional RNAi screen for HDMs and identified five potential HDMs essential for mouse ESC identity. In-depth analyses demonstrate that the closely related HDMs Jmjd2b and Jmjd2c are necessary for self-renewal of ESCs and induced pluripotent stem cell generation. Genome-wide occupancy studies reveal that Jmjd2b unique, Jmjd2c unique, and Jmjd2b-Jmjd2c common target sites belong to functionally separable Core, Polycomb repressive complex (PRC), and Myc regulatory modules, respectively. Jmjd2b and Nanog act through an interconnected regulatory loop, whereas Jmjd2c assists PRC2 in transcriptional repression. Thus, two HDMs of the same subclass exhibit distinct and combinatorial functions in control of the ESC state. Such complexity of HDM function reveals an aspect of multilayered transcriptional control.