Basic regulatory science behind drug substance and drug product specifications of monoclonal antibodies and other protein therapeutics

In this review,we focus on providing basics and examples for each component of the protein therapeutic specifications to interested pharmacists and biopharmaceutical scientists with a goal to strengthen understanding in regulatory science and compliance.Pharmaceutical specifications comprise a list of important quality attributes for testing,references to use for test procedures,and appropriate acceptance criteria for the tests,and they are set up to ensure that when a drug product is administered to a patient,its intended therapeutic benefits and safety can be rendered appropriately.Conformance of drug substance or drug product to the specifications is achieved by testing an article according to the listed tests and analytical methods and obtaining test results that meet the acceptance criteria.Quality attributes are chosen to be tested based on their quality risk,and consideration should be given to the merit of the analytical methods which are associated with the acceptance criteria of the specifications.Acceptance criteria are set forth primarily based on efficacy and safety profiles,with an increasing attention noted for patient-centric specifications.Discussed in this work are related guidelines that support the biopharmaceutical specification setting,how to set the acceptance criteria,and examples of the quality attributes and the analytical methods from 60 articles and 23 pharmacopeial monographs.Outlooks are also explored on process analytical technologies and other orthogonal tools which are on-trend in biopharmaceutical characterization and quality control.

Maillard reaction and immunogenicity of protein therapeutics

The recombinant DNA technology enabled the produ-ction of a variety of human therapeutic proteins. Accumulated clinical experience, however, indicates that the formation of antibodies against such proteins is a general phenomenon rather than an exception. The immunogenicity of therapeutic proteins results in inefficient therapy and in the development of undesired, sometimes life-threatening, side reactions. The human proteins, designed for clinical application, usually have the same amino acid sequence as their native prototypes and it is not yet fully clear what the reasons for their immunogenicity are. In previous studies we have demonstrated for the first time that interferon-b （IFN-b） pharmaceuticals, used for treatment of patients with multiple sclerosis, do contain advanced glycation end products （AGEs） that contribute to IFN-b immunogenicity. AGEs are the fnal products of a chemical reaction known as the Maillard reaction or glycation, which implication in protein drugs’ immunogenicity has been overlooked so far. Therefore, the aim of the present article is to provide a comprehensive overview on the Maillard reaction with emphasis on experimental data and theoretical consideration telling us why the Maillard reaction warrants special attention in the context of the well-documented protein drugs’ immunogenicity.

A phase separation-fortified bi-specific adaptor for conditional tumor killing

A common approach in therapeutic protein development involves employing synthetic ligands with multivalency,enabling sophisticated control of signal transduction.Leveraging the emerging concept of liquid-liquid phase separation(LLPS)and its ability to organize cell surface receptors into functional compartments,we herein have designed modular ligands with phase-separation modalities to engineer programmable interreceptor communications and precise control of signal pathways,thus inducing the rapid,potent,and specific apoptosis of tumor cells.Despite their simplicity,these“triggers”,named phase-separated Tumor Killers(hereafter referred to as psTK),are sufficient to yield interreceptor clustering of death receptors(represented by DR5)and tumor-associated receptors,with notable features:LLPSmediated robust high-order organization,well-choreographed conditional activation,and broad-spectrum capacity to potently induce apoptosis in tumor cells.The development of novel therapeutic proteins with phase-separation modalities showcases the power of spatially reorganizing signal transduction.This approach facilitates the diversification of cell fate and holds promising potential for targeted therapies against challenging tumors.

Supercritical fluid technology:A game-changer for biomacromolecular nanomedicine preparation and biomedical application

Biomacromolecules are attractive in biomedical applications as therapeutic agents and potential drug carriers due to their natural active components,good biocompatibility,and high targeting.However,their large relative molecular weight,complex structure,susceptibility to degradation,and poor stability limit their usefulness.Nanotechnology can address these issues by improving the therapeutic value,bioavailability,permeability,and absorption of biomacromolecules while regulating their retention time in the body.Especially,compelling evidence has been reported that supercritical fluid(SCF)technology has emerged as an alternative that maintains the integrity of biomacromolecules and reduces environmental contamination.In this review,we highlight a set of unique nanosizing strategies based on SCF technology for biomacromolecular nanomedicine,and extensively discuss their characteristics and mechanisms.In particular,the protein-based,nucleic acid-based,and polysaccharide-based nanomedicine preparations via SCF technology and their biomedical applications are summarized,and the potential for industrial production of biomacromolecular drugs is also considered.We further provide perspectives on the opportunities and challenges in this excellent field of biomacromolecular drugs nanotechnology.

Engineered protein nanodrug as an emerging therapeutic tool

Functional proteins are the most versatile macromolecules.They can be obtained by extraction from natural sources or by genetic engineering technologies.The outstanding selectivity,specificity,binding activity,and biocompatibility endow engineered proteins with outstanding performance for disease therapy.Nevertheless,their stability is dramatically impaired in blood circulation,hindering clinical translations.Thus,many strategies have been developed to improve the stability,efficacy,bioavailability,and productivity of therapeutic proteins for clinical applications.In this review,we summarize the recent progress in the fabrication and application of therapeutic proteins.We first introduce various strategies for improving therapeutic efficacy via bioengineering and nanoassembly.Furthermore,we highlight their diverse applications as growth factors,nanovaccines,antibody-based drugs,bioimaging molecules,and cytokine receptor antagonists.Finally,a summary and perspective for the future development of therapeutic proteins are presented.

Sol-gel synthesis,properties and protein loading/delivery capacity of hollow bioactive glass nanospheres with large hollow cavity and mesoporous shell

Hollow nanospheres exhibit unique properties and find a wide interest in several potential applications such as drug delivery.Herein,novel hollow bioactive glass nanospheres(HBGn)with large hollow cavity and large mesopores in their outer shells were synthesized by a simple and facile one-pot ultrasound assisted sol-gel method using PEG as the core soft-template.Interestingly,the produced HBGn exhibited large hollow cavity with ~43 nm in diameter and mesoporous shell of ~37 nm in thickness and 7 nm pore size along with nanosphere size around 117 nm.XPS confirmed the presence of Si and Ca elements at the surface of the HBGn outer shell.Notably,HBGn showed high protein loading capacity(~570 mg of Cyto c per 1 g of HBGn)in addition to controlled protein release over 5 d.HBGn also demonstrated a good in vitro capability of releasing calcium(Ca^(2+):170 ppm)and silicate(SiO_(4)^(4-):78 ppm)ions in an aqueous medium over 2 weeks under physiological-like conditions.Excellent in vitro growth of bone-like hydroxyapatite nanocrystals was exhibited by HBGn during the soaking in SBF.A possible underlying mechanism involving the formation of spherical aggregates(coils)of PEG was proposed for the formation process of HBGn.