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Protein stability against aggregation is a major quality concern for the production of safe and effective biopharmaceuticals. Amongst the different drivers of protein aggregation, increasing evidence indicates that interactions between proteins and interfaces represent a major risk factor for the formation of protein aggregates in aqueous solutions. Potentially harmful surfaces relevant to biologics manufacturing and storage include air-water and silicone oil-water interfaces as well as materials from different processing units, storage containers, and delivery devices. The impact of some of these surfaces, for instance originating from impurities, can be difficult to predict and control. Moreover, aggregate formation may additionally be complicated by the simultaneous presence of interfacial, hydrodynamic and mechanical stresses, whose contributions may be difficult to deconvolute. As a consequence, it remains difficult to identify the key chemical and physical determinants and define appropriate analytical methods to monitor and predict protein instability at these interfaces. In this review, we first discuss the main mechanisms of surface-induced protein aggregation. We then review the types of contact materials identified as potentially harmful or detected as potential triggers of proteinaceous particle formation in formulations and discuss proposed mitigation strategies. Finally, we present current methods to probe surface-induced instabilities, which represent a starting point towards assays that can be implemented in early-stage screening and formulation development of biologics., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest, (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Background: Functional tests for the diagnosis of heparin-induced thrombocytopenia (HIT) exhibit variable performance., Objectives: We evaluated in a multicenter study whether 5B9, a monoclonal anti-PF4/heparin IgG mimicking human HIT antibodies, could be used as an internal quality control., Methods: 5B9 was sent to 11 laboratories in seven countries, and six initial concentrations ranging from 10 to 400 μg/mL were tested by heparin-induced platelet activation assay (HIPA), serotonin release assay (SRA), platelet aggregation test (PAT), flow cytometry (FC), or heparin-induced multiple-electrode aggregometry (HIMEA). Each method was evaluated in three different laboratories using experimental procedures identical to those usually applied for the diagnosis of HIT by testing platelets from 10 different healthy donors., Results: The procedures used varied among the laboratories, particularly when platelet-rich plasma and whole blood were used. Nevertheless, positive results were obtained with at least 100 μg/ml of 5B9 for most donors tested by all centers (except one) performing HIPA, SRA, or HIMEA. FC and PAT results were more heterogeneous. FC results from one center that used washed platelets preincubated with PF4 were positive with all donors at 50 µg/ml 5B9, but at least 200 μg/ml of 5B9 were required to activate cells with most donors tested using PAT., Conclusion: This study confirms that HIT functional tests are not well standardized and exhibit variable sensitivity for the detection of platelet-activating antibodies. However, 5B9 is a potentially useful tool to standardize functional tests, to select responding platelet donors, and consequently to improve the performance of these assays and comparability between laboratories., (© 2021 International Society on Thrombosis and Haemostasis.)

The implementation of high-throughput sequencing (HTS) technologies in research and diagnostic laboratories has linked many new genes to rare bleeding, thrombotic, and platelet disorders (BTPD), and revealed multiple genetic variants linked to those disorders, many of them being of uncertain pathogenicity when considering the accepted evidence (variant consequence, frequency in control datasets, number of reported patients, prediction models, and functional assays). The sequencing effort has also resulted in resources for gathering disease-causing variants associated with specific genes, but for BTPD, such well-curated databases exist only for a few genes. On the other hand, submissions by individuals or diagnostic laboratories to the variant database ClinVar are hampered by the lack of a submission process tailored to capture the specific features of hemostatic diseases. As we move toward the implementation of HTS in the diagnosis of BTPD, the Scientific and Standardization Committee for Genetics in Thrombosis and Haemostasis has developed and tested a REDCap-based interface, aimed at the community, to submit curated genetic variants for diagnostic-grade BTPD genes. Here, we describe the use of the interface and the initial submission of 821 variants from 30 different centers covering 14 countries. This open-access variant resource will be shared with the community to improve variant classification and regular bulk data transfer to ClinVar., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

An increasing body of evidence shows that membraneless organelles are key components in cellular organization. These observations open a variety of outstanding questions about the physico-chemical rules underlying their assembly, disassembly and functions. Some molecular determinants of biomolecular condensates are challenging to probe and understand in complex in vivo systems. Minimalistic in vitro reconstitution approaches can fill this gap, mimicking key biological features, while maintaining sufficient simplicity to enable the analysis of fundamental aspects of biomolecular condensates. In this context, microfluidic technologies are highly attractive tools for the analysis of biomolecular phase transitions. In addition to enabling high-throughput measurements on small sample volumes, microfluidic tools provide for exquisite control of self-assembly in both time and space, leading to accurate quantitative analysis of biomolecular phase transitions. Here, with a specific focus on droplet-based microfluidics, we describe the advantages of microfluidic technology for the analysis of several aspects of phase separation. These include phase diagrams, dynamics of assembly and disassembly, rheological and surface properties, exchange of materials with the surrounding environment and the coupling between compartmentalization and biochemical reactions. We illustrate these concepts with selected examples, ranging from simple solutions of individual proteins to more complex mixtures of proteins and RNA, which represent synthetic models of biological membraneless organelles. Finally, we discuss how this technology may impact the bottom-up fabrication of synthetic artificial cells and for the development of synthetic protein materials in biotechnology., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

In the last decades, the pharmaceutical market has experienced an increase in the number of therapeutic proteins. The high activity and selectivity of these macromolecules is often achieved at the expense of complex structures, which exhibit several biophysical properties that must be carefully controlled and optimized for the successful development of these drugs as well as for guaranteeing their quality and safety. This need has motivated the application of a variety of biophysical techniques to analyze properties of therapeutic proteins and protein solutions including interactions, aggregation, solubility, viscosity, and thermal stability. After briefly summarizing currently available experimental approaches, we highlight the emerging possibilities offered by advances in microfluidic technology for the analysis of therapeutic proteins during manufacturing and formulation., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: N -methyl-d-aspartate receptors (NMDARs) contribute calcium influx in megakaryocytic cells but their roles remain unclear; both pro- and anti-differentiating effects have been shown in different contexts., Objectives: The aim of this study was to clarify NMDAR contribution to megakaryocytic differentiation in both normal and leukemic cells., Methods: Meg-01, Set-2, and K-562 leukemic cell lines were differentiated using phorbol-12-myristate-13-acetate (PMA, 10 nmol L -1 ) or valproic acid (VPA, 500 μmol L -1 ). Normal megakaryocytes were grown from mouse marrow-derived hematopoietic progenitors (lineage-negative and CD41a-enriched) in the presence of thrombopoietin (30-40 nmol L -1 ). Marrow explants were used to monitor proplatelet formation in the native bone marrow milieu. In all culture systems, NMDARs were inhibited using memantine and MK-801 (100 μmol L -1 ); their effects compared against appropriate controls., Results: The most striking observation from our studies was that NMDAR antagonists markedly inhibited proplatelet formation in all primary cultures employed. Proplatelets were either absent (in the presence of memantine) or short, broad and intertwined (with MK-801). Earlier steps of megakaryocytic differentiation (acquisition of CD41a and nuclear ploidy) were maintained, albeit reduced. In contrast, in leukemic Meg-01 cells, NMDAR antagonists inhibited differentiation in the presence of PMA and VPA but induced differentiation when applied by themselves., Conclusions: NMDAR-mediated calcium influx is required for normal megakaryocytic differentiation, in particular proplatelet formation. However, in leukemic cells, the main NMDAR role is to inhibit differentiation, suggesting diversion of NMDAR activity to support leukemia growth. Further elucidation of the NMDAR and calcium pathways in megakaryocytic cells may suggest novel ways to modulate abnormal megakaryopoiesis.

Hemizygous deletion of a variable region on chromosome 11q containing FLI1 causes an inherited platelet-related bleeding disorder in Paris-Trousseau thrombocytopenia and Jacobsen syndrome. These multisystem disorders are also characterized by heart anomalies, changes in facial structure, and intellectual disability. We have identified a consanguineous family with autosomal recessive inheritance of a bleeding disorder that mimics Paris-Trousseau thrombocytopenia but has no other features of the 11q23 deletion syndrome. Affected individuals in this family have moderate thrombocytopenia; absent collagen-induced platelet aggregation; and large, fused α-granules in 1% to 5% of circulating platelets. This phenotype was caused by a FLI1 homozygous c.970C>T-point mutation that predicts an arginine-to-tryptophan substitution in the conserved ETS DNA-binding domain of FLI1. This mutation caused a transcription defect at the promoter of known FLI1 target genes GP6, GP9, and ITGA2B, as measured by luciferase assay in HEK293 cells, and decreased the expression of these target proteins in affected members of the family as measured by Western blotting of platelet lysates. This kindred suggests abnormalities in FLI1 as causative of Paris-Trousseau thrombocytopenia and confirms the important role of FLI1 in normal platelet development., (© 2015 by The American Society of Hematology.)