Recent Advances in Biologic Therapeutic N-Glycan Preparation Techniques and Analytical Methods for Facilitating Biomanufacturing Automation.

N-glycosylation is a post-translational modification that occurs during the production of monoclonal antibody (mAb) therapeutics. During production of mAb based therapeutics the use of various hosts and cell culture additives attribute to glycan heterogeneity. The safety and efficacy of monoclonal antibodies with mechanism of actions that utilize Fc effector functions can be negatively impacted by glycan heterogeneity and thus is often considered a critical quality attribute (CQA). In this mini review, we discuss recent advances in mAb sample preparation specifically focused on denaturation, enzymatic processing, and released glycans derivatization methods. Additionally, we review the recent advances in characterization of released and intact N-glycans using chromatography, capillary electrophoresis, and mass spectrometry techniques with a focus on rapid, automated approaches that support analysis of glycosylation profiles of biopharmaceuticals. We delve into advances within sample preparation techniques that allow for rapid and robust sample preparation as well as how these techniques are being used for innovative at-line high-throughput screening and process analytical technology (PAT). The future of biomanufacturing is focused on decreasing process costs while increasing process understanding and quality for novel biologic candidates and biosimilars. Therefore, advances in PAT for biotherapeutics will positively influence current manufacturing practices and enable further bioprocess automation.

Defining the hydrophobic interactions that drive competence stimulating peptide (CSP)-ComD binding in Streptococcus pneumoniae.

Quorum sensing (QS) is a cell-cell communication mechanism that enables bacteria to assess their population density and alter their behavior upon reaching high cell number. Many bacterial pathogens utilize QS to initiate an attack on their host, thus QS has attracted significant attention as a potential antivirulence alternative to traditional antibiotics. Streptococcus pneumoniae, a notorious human pathogen responsible for a variety of acute and chronic infections, utilizes the competence regulon and its associated signaling peptide, the competence stimulating peptide (CSP), to acquire antibiotic resistance and establish an infection. In this work, we sought to define the binding pockets within the ComD1 receptor used for binding the hydrophobic side-chain residues in CSP1 through the introduction of highly-conservative point mutations within the peptide. Optimization of these binding interactions could lead to the development of highly potent CSP-based QS modulators while the inclusion of non-natural amino acids within the CSP sequence would confer resistance to protease degradation, a requirement for drug candidates.