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.

Online monitoring and control of upstream cell culture process using 1D and 2D-LC with SegFlow interface.

The biopharmaceutical industry is transitioning from currently deployed batch-mode bioprocessing to a highly efficient and agile next-generation bioprocessing with the adaptation of continuous bioprocessing, which reduces capital investment and operational costs. Continuous bioprocessing, aligned with FDA's quality-by-design platform, is designed to develop robust processes to deliver safe and effective drugs. With the deployment of knowledge-based operations, product quality can be built into the process to achieve desired critical quality attributes (CQAs) with reduced variability. To facilitate next-generation continuous bioprocessing, it is essential to embrace a fundamental shift-in-paradigm from "quality-by-testing" to "quality-by-design," which requires the deployment of process analytical technologies (PAT). With the adaptation of PAT, a systematic approach of process and product understanding and timely process control are feasible. Deployment of PAT tools for real-time monitoring of CQAs and feedback control is critical for continuous bioprocessing. Given the current deficiency in PAT tools to support continuous bioprocessing, we have integrated Infinity 2D-LC with a post-flow-splitter in conjunction with the SegFlow autosampler to the bioreactors. With this integrated system, we have established a platform for online measurements of titer and CQAs of monoclonal antibodies as well as amino acid analysis of bioreactor cell culture.

Systematic Development of a Size Exclusion Chromatography Method for a Monoclonal Antibody with High Surface Aggregation Propensity (SAP) Index.

Size Exclusion Chromatography (SEC) has been widely used to assess aggregate content in bio-pharmaceutical drugs such as monoclonal antibodies (mAbs), and is routinely used during method development and release testing. Electrostatic interactions between protein analytes and SEC column resin are commonly observed besides the primary mode of size separation during SEC method development, which needs to be minimized. An effective method to minimize electrostatic interactions is through increasing mobile phase (MP) salt concentration. However; increasing salt concentration in MP will induce increased hydrophobicity of proteins and increased hydrophobic interactions between protein and stationary phase, as demonstrated for mAb-A in this paper, a protein with high surface aggregation propensity (SAP) score and an isoelectric point near mobile phase pH. In this work, a systematic, Design of Experimental approach was taken to identify optimal SEC method conditions including column type, buffer composition, ionic strength, pH and additives. The optimized method was demonstrated to be robust towards small changes in method operation conditions and was qualified for use in product release and stability studies. Additionally, biophysical and computational studies were performed to elucidate the role of MP additives, which supports the use of arginine as an essential additive to minimize undesirable hydrophobic interactions between proteins and stationary phase.

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.

Streamlining the polishing step development process via physicochemical characterization of monoclonal antibody aggregates.

When developing purification processes for monoclonal antibodies (mAbs), ensuring the effective removal of high molecular weight (HMW) species is often challenging and labor intensive. In this work, we present a bottom-up characterization approach to achieve streamlined polishing step development as well as a more fundamental understanding of the protein of interest. Prior to physicochemical characterization, in-process HMW species of two IgG4 mAbs (mAb A and mAb B) were isolated via semi-preparative size exclusion chromatography (SEC). Key differences in approximate molecular weight, net charge, and native surface hydrophobicity were then identified using multi-angle light scattering (SEC-MALS), analytical-scale chromatographic screening, isoelectric focusing, and structural aggregation propensity modeling. SEC-MALS revealed two main HMW isoforms for each mAb: dimers and 1.7-mers for mAb A, and tetramers and dimers for mAb B. Analytical-scale chromatographic screening showed promising trends in charge-based separation for mAb A, and hydrophobic-based separation for mAb B. Isoelectric focusing data detected a 30% increase in acidic variants for mAb A HMW species relative to monomer, and a 20% increase in basic variants for mAb B HMW species. Lastly, analytical-scale characterization data was successfully applied to preparative scale purification conditions, producing results highly similar to those observed during analytical characterization of the isolated species. By using this high-throughput approach as a template for preparative-scale process development, key physicochemical differences between aggregate and monomer species were utilized to determine optimal polishing steps for HMW removal.

Titration of ionizable groups in proteins using multiple neutron data sets from a single crystal: application to the small GTPase Ras.

Neutron protein crystallography (NPC) reveals the three-dimensional structures of proteins, including the positions of H atoms. The technique is particularly suited to elucidate ambiguous catalytic steps in complex biochemical reactions. While NPC uniquely complements biochemical assays and X-ray structural analyses by revealing the protonation states of ionizable groups at and around the active site of enzymes, the technique suffers from a major drawback: large single crystals must be grown to compensate for the relatively low flux of neutron beams. However, in addition to revealing the positions of hydrogens involved in enzyme catalysis, NPC has the advantage over X-ray crystallography that the crystals do not suffer radiation damage. The lack of radiation damage can be exploited to conduct in crystallo parametric studies. Here, the use of a single crystal of the small GTPase Ras to collect three neutron data sets at pD 8.4, 9.0 and 9.4 is reported, enabling an in crystallo titration study using NPC. In addition to revealing the behavior of titratable groups in the active site, the data sets will allow the analysis of allosteric water-mediated communication networks across the molecule, particularly regarding Cys118 and three tyrosine residues central to these networks, Tyr32, Tyr96 and Tyr137, with pKa values expected to be in the range sampled in our experiments.

An engineered protein antagonist of K-Ras/B-Raf interaction.

Ras is at the hub of signal transduction pathways controlling cell proliferation and survival. Its mutants, present in about 30% of human cancers, are major drivers of oncogenesis and render tumors unresponsive to standard therapies. Here we report the engineering of a protein scaffold for preferential binding to K-Ras G12D. This is the first reported inhibitor to achieve nanomolar affinity while exhibiting specificity for mutant over wild type (WT) K-Ras. Crystal structures of the protein R11.1.6 in complex with K-Ras WT and K-Ras G12D offer insight into the structural basis for specificity, highlighting differences in the switch I conformation as the major defining element in the higher affinity interaction. R11.1.6 directly blocks interaction with Raf and reduces signaling through the Raf/MEK/ERK pathway. Our results support greater consideration of the state of switch I and provide a novel tool to study Ras biology. Most importantly, this work makes an unprecedented contribution to Ras research in inhibitor development strategy by revealing details of a targetable binding surface. Unlike the polar interfaces found for Ras/effector interactions, the K-Ras/R11.1.6 complex reveals an extensive hydrophobic interface that can serve as a template to advance the development of high affinity, non-covalent inhibitors of K-Ras oncogenic mutants.

The small GTPases K-Ras, N-Ras, and H-Ras have distinct biochemical properties determined by allosteric effects.

H-Ras, K-Ras, and N-Ras are small GTPases that are important in the control of cell proliferation, differentiation, and survival, and their mutants occur frequently in human cancers. The G-domain, which catalyzes GTP hydrolysis and mediates downstream signaling, is 95% conserved between the Ras isoforms. Because of their very high sequence identity, biochemical studies done on H-Ras have been considered representative of all three Ras proteins. We show here that this is not a valid assumption. Using enzyme kinetic assays under identical conditions, we observed clear differences between the three isoforms in intrinsic catalysis of GTP by Ras in the absence and presence of the Ras-binding domain (RBD) of the c-Raf kinase protein (Raf-RBD). Given their identical active sites, isoform G-domain differences must be allosteric in origin, due to remote isoform-specific residues that affect conformational states. We present the crystal structure of N-Ras bound to a GTP analogue and interpret the kinetic data in terms of structural features specific for H-, K-, and N-Ras.

Neutron Crystal Structure of RAS GTPase Puts in Question the Protonation State of the GTP γ-Phosphate.

RAS GTPase is a prototype for nucleotide-binding proteins that function by cycling between GTP and GDP, with hydrogen atoms playing an important role in the GTP hydrolysis mechanism. It is one of the most well studied proteins in the superfamily of small GTPases, which has representatives in a wide range of cellular functions. These proteins share a GTP-binding pocket with highly conserved motifs that promote hydrolysis to GDP. The neutron crystal structure of RAS presented here strongly supports a protonated γ-phosphate at physiological pH. This counters the notion that the phosphate groups of GTP are fully deprotonated at the start of the hydrolysis reaction, which has colored the interpretation of experimental and computational data in studies of the hydrolysis mechanism. The neutron crystal structure presented here puts in question our understanding of the pre-catalytic state associated with the hydrolysis reaction central to the function of RAS and other GTPases.