Two-dimensional size exclusion reversed-phase liquid chromatography for quantitative analysis of L1 proteins in complex vaccine matrices.

The quantitation of the major capsid protein L1 is an important metric during the pharmaceutical manufacturing of human papilloma virus (HPV) vaccines, as they are critical components of virus like particles (VLPs) that form the core of the drug product. During the production of VLPs, the L1 protein is present in multiple states, including monomer, multimer, fully formed VLPs and aggregate species, whose expression levels provides an important read-out of upstream productivity and downstream purification efficiency through the measurement of step yields. However, quantitation of total L1 protein is challenging not only due to its presence in multiple states, but also due to the matrix complexity and purification stage of the samples, which spans complex cell lysate to cleaner post purification material.

Two-Dimensional SEC-SEC-UV-MALS-dRI Workflow for Streamlined Analysis and Characterization of Biopharmaceuticals.

The emergence of complex biological modalities in the biopharmaceutical industry entails a significant expansion of the current analytical toolbox to address the need to deploy meaningful and reliable assays at an unprecedented pace. Size exclusion chromatography (SEC) is an industry standard technique for protein separation and analysis. Some constraints of traditional SEC stem from its restricted ability to resolve complex mixtures and notoriously long run times while also requiring multiple offline separation conditions on different pore size columns to cover a wider molecular size distribution.

Pioneering Just-in-Time (JIT) Strategy for Accelerating Raman Method Development and Implementation for Biologic Continuous Manufacturing.

Raman spectroscopy is a popular process analytical technology (PAT) tool that has been increasingly used to monitor and control the monoclonal antibody (mAb) manufacturing process. Although it allows the characterization of a variety of quality attributes by developing chemometric models, a large quantity of representative data is required, and hence, the model development process can be time-consuming. In recent years, the pharmaceutical industry has been expediting new drug development in order to achieve faster delivery of life-changing drugs to patients.

Automated Online-Sampling Multidimensional Liquid Chromatography with Feedback-Control Capability as a Framework for Real-Time Monitoring of mAb Critical Quality Attributes in Multiple Bioreactors.

Real-time monitoring of biopharmaceutical reactors is becoming increasingly important as the processes become more complex. During the continuous manufacturing of monoclonal antibodies (mAbs), the desired mAb product is continually created and collected over a 30 day process, where there can be changes in quality over that time. Liquid chromatography (LC) is the workhorse instrumentation capable of measuring mAb concentration as well as quality attributes such as aggregation, charge variants, oxidation, etc.

Biosynthetic Origin of Formylaminooxyvinylglycine and Characterization of the Formyltransferase GvgI.

4-Formylaminooxyvinylglycine (FVG) is an herbicidal and antibacterial nonproteinogenic amino acid produced by several strains of the species complex. It contains a unique vinyl alkoxyamine moiety with an O-N bond, and its biosynthetic origin remains unknown. Here, we show that the cluster from WH6 is responsible for the biosynthesis of FVG and two additional O-N bond-containing oxyvinylglycines, guanidinooxyvinylglycine and aminooxyvinylglycine.

Fiber-based HIC capture loop for coupling of protein A and size exclusion chromatography in a two-dimensional separation of monoclonal antibodies.

During process development and manufacturing of monoclonal antibodies (mAbs), it is critical to characterize structure-function relationships to properly control the levels of mAb aggregation and potency of the protein product. With two-dimensional high performance liquid chromatography (2D-HPLC) technology, protein A (ProA) affinity chromatography can be used in the first dimension to isolate and measure the concentration of mAb, with the effluent transferred to a second dimension of size exclusion chromatography (SEC) to measure purity (i.e.

Rapid two-dimensional Protein-A size exclusion chromatography of monoclonal antibodies for titer and aggregation measurements from harvested cell culture fluid samples.

The success of monoclonal antibody (mAb) therapeutics have increased pharmaceutical investment in mAb production, which has led to a greater demand of technologies to efficiently characterize these biotherapeutics. The large size and heterogeneity of mAbs require the measurement of multiple critical quality attributes (CQAs) during production. The current workflow to measure CQAs of antibodies involves multiple one-dimensional liquid chromatography methods, including Protein-A (ProA), ion-exchange (IEX), reversed-phase, size exclusion (SEC), hydrophilic interaction, and hydrophobic interaction (HIC).

Discovery and Characterization of FMN-Binding β-Glucuronidases in the Human Gut Microbiome.

The human gut microbiota encodes β-glucuronidases (GUSs) that play key roles in health and disease via the metabolism of glucuronate-containing carbohydrates and drugs. Hundreds of putative bacterial GUS enzymes have been identified by metagenomic analysis of the human gut microbiome, but less than 10% have characterized structures and functions. Here we describe a set of unique gut microbial GUS enzymes that bind flavin mononucleotide (FMN).

Gut Microbial β-Glucuronidase Inhibition via Catalytic Cycle Interception.

Microbial β-glucuronidases (GUSs) cause severe gut toxicities that limit the efficacy of cancer drugs and other therapeutics. Selective inhibitors of bacterial GUS have been shown to alleviate these side effects. Using structural and chemical biology, mass spectrometry, and cell-based assays, we establish that piperazine-containing GUS inhibitors intercept the glycosyl-enzyme catalytic intermediate of these retaining glycosyl hydrolases.

In Vitro Biosynthesis of the Nonproteinogenic Amino Acid Methoxyvinylglycine.

Oxyvinylglycines are a family of nonproteinogenic amino acids featuring an essential vinyl ether conferring mechanism-based inhibition of pyridoxal phosphate enzymes. The gene clusters for a few oxyvinylglycines are known, yet the biosynthetic origin of the vinyl ether is elusive. The in vitro biosynthesis of methoxyvinylglycine or l-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is reported.

Enzymatic basis of "hybridity" in thiomarinol biosynthesis.

Thiomarinol is a naturally occurring double-headed antibiotic that is highly potent against methicillin-resistant Staphylococcus aureus. Its structure comprises two antimicrobial subcomponents, pseudomonic acid analogue and holothin, linked by an amide bond. TmlU was thought to be the sole enzyme responsible for this amide-bond formation.