Explore the vast range of titles available.

Isomerization of aspartic acid (Asp) in therapeutic proteins could lead to safety and efficacy concerns. Thus, accurate quantitation of various Asp isomerization along with kinetic understanding of the variant formations is needed to ensure optimal process development and sufficient product quality control. In this study, we first observed Asp-succinimide conversion in complementarity-determining regions (CDRs) Asp-Gly motif of a recombinant mAb through ion exchange chromatography, intact protein analysis by mass spectrometry, and LC-MS/MS. Then, we developed a specific peptide mapping method, with optimized sample digestion conditions, to accurately quantitate Asp-succinimide-isoAsp variants at peptide level without method-induced isomerization. Various kinetics of Asp-succinimide-isoAsp isomerization pathways were elucidated using 18O labeling followed by LC-MS analysis. Molecular modeling and molecular dynamic simulation provide additional insight on the kinetics of Asp-succinimide formation and stability of succinimide intermediate. Findings of this work shed light on the molecular construct and the kinetics of the formation of isoAsp and succinimide in peptides and proteins, which facilitates analytical method development, protein engineering, and late phase development for commercialization of therapeutic proteins.

icIEF (imaged capillary isoelectric focusing) charge variants characterization of biologics is important for understanding product charge isoforms and how they affect product quality. Protein post translational modifications (PTMs), such as deamidation, sialylation, glycation, c-terminal lysine cleavage and c-terminal glycine amidation, are common sources of acidic and basic charge variants of proteins and antibodies. Our PEGylated protein was expressed in E. coli which significantly reduced the PTMs, only deamidation and carbamylation were observed as charge variants related PTMs by LC–MS peptide mapping analysis. Deamidation introduces negative charge and was experimentally confirmed to be the major contributor of the acidic peaks in icIEF charge profiling of our PEGylated protein. The possible PTMs for basic variants were screened, but they were not observed by LC–MS peptide mapping. Beyond PTM induced charge heterogeneity, we found that N terminal methionine excision (NME) and residual un-PEGylated protein contributed to the basic peaks. NME is common in E. coli expression which is often used for PEGylated proteins; thus, it could be leveraged to charge variants characterization of other PEGylated proteins. Our finding of residual un-PEGylated protein contributing to basic peaks is being reported for the first time and could broaden the knowledge of possible charge variants in PEGylated proteins.

This study emphasizes the critical importance of closely monitoring and controlling the sialic acid content in therapeutic glycoproteins, including EPO, interferon‐γ, Orencia, Enbrel, and others, as the level of sialylation directly impacts their pharmacokinetics (PK), immunogenicity, potency, and overall clinical performance due to its influence on protein clearance via hepatic asialoglycoprotein receptors (ASGPR). The ASGPR recognizes and binds to glycoproteins exposed to terminal galactose or N‐acetylgalactosamine residues, leading to receptor‐mediated endocytosis. Recent studies have demonstrated that sialylation of O‐linked glycan plays a role in protecting against macrophage galactose lectin (MGL)‐mediated clearance. In addition to the impact on serum half‐life, sialylation can influence other clinical outcomes, including immunogenicity, potency, and cytotoxicity. Therefore, the level of sialic acid is a critical quality attribute (CQA), and monitoring and regulating sialylation has become a regulatory requirement to ensure desired clinical performance. To achieve consistent levels of sialic acid‐to‐protein ratio, the time of upstream harvest and conductivity of downstream wash buffers must be tightly regulated based on the sialic acid content. Therefore, the utilization of process analytical technology (PAT) tools for generating real‐time or near‐real‐time sialic acid content is a business‐critical requirement. This work demonstrates the utility of an integrated PAT system for near real‐time online sialic acid measurements. The system consists of a micro‐sequential injection analyzer (µSIA) interfaced with SegFlow and an ultra performance liquid chromatography (UPLC). The fully automated architecture exemplifies the execution of online sampling, automatic sample preparation, and subsequent online UPLC analysis. This carefully orchestrated PAT framework effectively supports the requirements of QbD‐driven continuous bioprocessing.

The aim of the study is to solve a significant challenge of extending the half-life of therapeutic proteins using crystalline biopharmaceuticals and without redesigning the molecules. Crystals of recombinant human growth hormone were coated with a monomolecular layer of positively charged poly(arginine). The pharmacokinetics and pharmacodynamics of this poly(arginine)-coated human growth hormone crystalline formulation were determined in hypophysectomized rats and monkeys. Here we have demonstrated for the first time that crystals of human growth hormone coated with positively charged poly(arginine) allowed for in vivo pharmacokinetic release profiles of over several days in animal models. The efficacy of this crystalline formulation injected subcutaneously once a week was found to be equivalent to seven daily soluble injections in the standard weight gain assay using the hypophysectomized rat model and in measurement of serum insulin-like growth factor in monkeys. The nonviscous nature of the suspension facilitated easy administration through a fine, 30-gauge needle and should provide for improved patient convenience and compliance. The approach described here offers an exciting possibility of being broadly applicable to other therapeutic proteins.

The global market for both pharmaceutical and bio-pharmaceutical drugs combined has reached $825 billion in yearly sales. As generic companies can easily duplicate pharmaceutical drugs like aspirin with simple structures, patent expired pharmaceutical drugs can be marketed at a price discount of 70%. However, it is impossible for the bio-generic companies to duplicate the biopharmaceutical drugs identical to the originator’s drug. Because the two biopharmaceutical drugs are not identical, expensive and lengthy clinical trials are required to ensure safety. Therefore, the bio-generic companies cannot offer significant price discounts. The problem addressed in this qualitative exploratory study is the prevalence of high bio-pharmaceutical drug prices attributed to the absence of a business process for manufacturing bio-pharmaceutical drugs efficiently. Recent introduction of bio-generic legislation through Food and Drug Administration (FDA) to alleviate the high price of biopharmaceutical drugs has been ineffective due to the difficulty of demonstrating its comparability to the originator’s drug without conducting clinical trials. Hence, the bio-generic introduction strategy is not expected to attain a significant drug price reduction. The purpose of this study is to propose a hypothetical Business Process Management (BPM) model with the integration of emerging process technologies to overcome the current inefficiencies. The prospect of integrating various emerging technologies to improve the efficiency of biopharmaceutical manufacturing processes was evaluated through interviewing subject matter experts in the biopharmaceutical sector in which the interview candidates were recruited by posting an interview participation request form on Labroots website, a leading social networking site targeted for scientific, medical and academic communities.