Recent advances in induced proximity modalities

Challenging disease targets necessitate new approaches for therapeutic intervention. Rewiring protein-biomolecule interactions with proximity-inducing agents extends intervention opportunities beyond target agonism or inhibition. Spanning varied molecular phenotypes and diverse target classes, proximity-inducing agents demonstrate immense potential across target degradation, cleavage, and post-translational editing. Here, we review a selection of exciting developments in the concepts and mechanisms of induced proximity-driven strategies from the last two years. Key technological advances that enable these discoveries and expand the scope of targets and machinery for induced-proximity modalities are highlighted.     

耐甲氧西林金黄色葡萄球菌感染控制策略的新进展

耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus Aureus, MRSA)已成为一种越来越具有侵袭性和流行性的病原菌。通过染色体介导、质粒转移、基因表达调控和主动外排系统等途径,MRSA对包括万古霉素在内的多种抗生素产生了抗药性。从生态学和进化的角度来考虑,仅仅通过利用抗生素本身来解决耐药性的困境是不够的。这就迫使人们去寻找一类完全不同于化学药物治疗MRSA感染的机制。随着对免疫学和生物学认识的不断深入,基于免疫逃逸、细菌群体感应、基因调控等理论的发现,涌现了一批生物制剂抗MRSA感染的研究,相对于传统的抗生素治疗这是一个全新突破的领域。此外还有传统中草药的研发也提示其在抗MRSA方面存在积极的活力。本综述总结了生物制剂、新型策略化学药物和传统中草药治疗MRSA感染的最新进展,以寻找解决抗生素治疗困境的新线索。     

Influence of molecular size on tissue distribution of antibody fragments

Biodistribution coefficients (BC) allow estimation of the tissue concentrations of proteins based on the plasma pharmacokinetics. We have previously established the BC values for monoclonal antibodies. Here, this concept is extended by development of a relationship between protein size and BC values. The relationship was built by deriving the BC values for various antibody fragments of known molecular weight from published biodistribution studies. We found that there exists a simple exponential relationship between molecular weight and BC values that allows the prediction of tissue distribution of proteins based on molecular weight alone. The relationship was validated by a priori predicting BC values of 4 antibody fragments that were not used in building the relationship. The relationship was also used to derive BC50 values for all the tissues, which is the molecular weight increase that would result in 50% reduction in tissue uptake of a protein. The BC50 values for most tissues were found to be ~35 kDa. An ability to estimate tissue distribution of antibody fragments based on the BC vs. molecular size relationship established here may allow better understanding of the biologics concentrations in tissues responsible for efficacy or toxicity. This relationship can also be applied for rational development of new biotherapeutic modalities with optimal biodistribution properties to target (or avoid) specific tissues.     

Comparative analysis of amino acid metabolism and transport in CHO variants with different levels of productivity

Chinese hamster ovary (CHO) cells are widely used for the production of biopharmaceuticals; however, our understanding of several physiological elements that contribute to productivity is limited. One of these is amino acid transport and how its limitation and/or regulation might affect productivity. To further our understanding, we have examined the expression of 40 mammalian amino acid transporter genes during batch cultures of three CHO cell lines: a non-producer and two antibody-producing cell lines with different levels of productivity. In parallel, extracellular and intracellular levels of amino acids were quantified. The aim was to identify differences in gene regulation between cell lines and within culture. Our results show that three transporters associated with transport of taurine and β-alanine, acidic amino acids and branched chain amino acids, are highly upregulated in both antibody-producing cell lines but not in the non-producer. Additionally, genes associated with the transport of amino acids related to the glutathione pathway (alanine, cysteine, cystine, glycine, glutamate) were found to be highly upregulated during the stationary phase of cell culture, correlating well with literature data on the importance of the pathway. Our analysis highlights potential markers for cell line selection and targets for process optimization.     

Advances and challenges in analytical characterization of biotechnology products: mass spectrometry-based approaches to study properties and behavior of protein therapeutics

Biopharmaceuticals are a unique class of medicines due to their extreme structural complexity. The structure of these therapeutic proteins is critically important for their efficacy and safety, and the ability to characterize it at various levels (from sequence to conformation) is critical not only at the quality control stage, but also throughout the discovery and design stages. Biological mass spectrometry (MS) offers a variety of approaches to study structure and behavior of complex protein drugs and has already become a default tool for characterizing the covalent structure of protein therapeutics, including sequence and post-translational modifications. Recently, MS-based methods have also begun enjoying a dramatic growth in popularity as a means to provide information on higher order structure and dynamics of biotechnology products. In particular, hydrogen/deuterium exchange MS and charge state distribution analysis of protein ions in electrospray ionization (ESI) MS offer a convenient way to assess the integrity of protein conformation. Native ESI MS also allows the interactions of protein drugs with their therapeutic targets and other physiological partners to be monitored using simple model systems. MS-based methods are also applied to study pharmacokinetics of biopharmaceutical products, where they begin to rival traditional immunoassays. MS already provides valuable support to all stages of development of biopharmaceuticals, from discovery to post-approval monitoring, and its impact on the field of biopharmaceutical analysis will undoubtedly continue to grow.     

Monoclonal antibody higher order structure analysis by high throughput protein conformational array

The elucidation of antibody higher order structure (HOS) is critical in therapeutic antibody development. Since HOS determines the protein bioactivity and chemo-physical properties, this knowledge can help to ensure that the safety and efficacy attributes are not compromised. Protein conformational array (PCA) is a novel method for determining the HOS of monoclonal antibodies. Previously, we successfully utilized an enzyme-linked immunosorbent assay (ELISA)-based PCA along with other bioanalytical tools to elucidate the structures of antibody aggregates. In this study, applying a new multiplex-based PCA with 48-fold higher throughput than the ELISA-based one we revealed structural differences between different antibody molecules and antibody structure changes affected by various processing conditions. The PCA analysis of antibody molecules clearly demonstrated significant differences between IgG1 and IgG4 subclasses in epitope exposure and folding status. Furthermore, we applied small angle X-ray scattering to decipher mechanistic insights of PCA technology and validate structural information obtained using PCA. These findings enhance our fundamental understanding of mAbs' HOS in general. The PCA analysis of antibody samples from various processing conditions also revealed that antibody aggregation caused significantly higher exposure of antibody epitopes, which potentially led to a “foreign” molecule that could cause immunogenicity. The PCA data correlated well with protein stability results from traditional methods such as size-exclusion chromatography and protein thermal shift assay. Our study demonstrated that high throughput PCA is a suitable method for HOS analysis in the discovery and development of therapeutic antibodies.     

蛋白质类生物制品几种不合格冻干外观及解决方案

冻干即真空冷冻干燥,是利用升华的原理使物料脱水的一种干燥技术,其广泛用于生物制品,特别是蛋白质类生物制品的生产,以保护生物制品生物活性成分。外观是冻干制品的重要质量属性之一,合格的冻干制品外观应是疏松多孔、色泽均匀、质地细腻的固体。在大规模生产中,由于冻干参数复杂、设备操作的灵活性等因素,冻干制品的饼块有时会出现收缩、裂化、塌陷、雾化、回熔、喷瓶、结膜等不合格外观,从而造成极大的经济损失。现就近年来在蛋白质类生物制品冻干过程中易发生的几种不合格外观及解决方案作一概述。     

Review on the worldwide regulatory framework for biosimilars focusing on the Mexican case as an emerging market in Latin America

The global biopharmaceutical market is worth over $100 billion USD. Nearly 90% of these products will lose their patent in the next ten years, leading to the commercialization of their subsequent versions, known as ‘biosimilars’. Biosimilars are much more complex molecules than chemically synthesized generics in terms of size, structure, stability, microheterogeneity, manufacture, etc. Therefore, a specific regulatory framework is needed in order to demonstrate their comparability with innovative products, as well as their quality, safety and efficacy. The EU published the first regulatory pathway in 2005 and has approved 14 biosimilars. Mexico has recently developed a clear regulatory pathway for these products. Their legal basis was established in Article 222 Bis of General Law of Health in 2009, clear specifications in the Regulation for Health Goods in 2011, and further requirements in the Mexican Official Norm NOM-EM-001-SSA1-2012. The aim of this review is to summarize the regulatory pathways for biosimilars in the world with a special focus on Mexican experience, so as contribute to the development of regulations in other countries.