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1.
Int J Toxicol ; 41(4): 291-296, 2022 08.
Article in English | MEDLINE | ID: mdl-35656559

ABSTRACT

The IQ Consortium NHP Reuse Working Group (WG) comprises members from 15 pharmaceutical and biotechnology companies. In 2020, the WG developed and distributed a detailed questionnaire on protein non-naïve NHP reuse to the WG member companies. The WG received responses from key stakeholders including principal investigators, facility managers, animal welfare officers and research scientists. This paper's content reflects the consolidated opinion of the WG members and the questionnaire responses on the subject of NHP reuse within nonclinical programs at all stages of research and development. Many of the pharmaceutical companies represented in the working group or participating in the questionnaire have already achieved some level of NHP reuse in their nonclinical programs, but the survey results suggested that there is significant potential to increase NHP reuse further and a need to understand the considerations involved in reuse more clearly. The WG has also focused carefully on the inherent concerns and risks of implementing protein non-naive NHP reuse and has evaluated the best methods of risk assessment and decision-making. This paper presents a discussion on the challenges and opportunities surrounding protein non-naïve NHP reuse and aims to stimulate further industry dialogue on the subject and provide guidance for pharmaceutical companies to establish roadmaps and decision trees enabling increased protein non-naïve NHP reuse. In addition, this paper represents a solid basis for collaborative engagement between pharmaceutical and biotechnology companies with contract research organizations (CROs) to discuss how the availability of protein non-naïve NHP within CROs can be better leveraged for their use within nonclinical studies.


Subject(s)
Drug Discovery , Primates , Animals , Drug Evaluation, Preclinical/methods , Drug Industry/methods , Pharmaceutical Preparations
2.
AAPS J ; 24(3): 68, 2022 05 12.
Article in English | MEDLINE | ID: mdl-35554731

ABSTRACT

The determination of a tailored anti-drug antibody (ADA) testing strategy is based on the immunogenicity risk assessment to allow a correlation of ADAs with changes to pharmacokinetics, efficacy, and safety. The clinical impact of ADA formation refines the immunogenicity risk assessment and defines appropriate risk mitigation strategies. Health agencies request for high-risk biotherapeutics to extend ADA monitoring for patients that developed an ADA response to the drug until ADAs return to baseline levels. However, there is no common understanding in which cases an extension of ADA follow-up sampling beyond the end of study (EOS) defined in the clinical study protocol is required. Here, the Immunogenicity Strategy Working Group of the European Immunogenicity Platform (EIP) provides recommendations on requirements for an extension of ADA follow-up sampling in clinical studies where there is a high risk of serious consequences from ADAs. The importance of ADA evaluation during a treatment-free period is recognized but the decision whether to extend ADA monitoring at a predefined EOS should be based on evaluation of ADA data in the context of corresponding clinical signals. If the clinical data set shows that safety consequences are minor, mitigated, or resolved, further ADA monitoring may not be required despite potentially detectable ADAs above baseline. Extended ADA monitoring should be centered on individual patient benefit.


Subject(s)
Antibodies , Humans
3.
Chem Sci ; 11(27): 7031-7039, 2020 Jun 17.
Article in English | MEDLINE | ID: mdl-34122996

ABSTRACT

The mechanism of amyloid co-aggregation and its nucleation process are not fully understood in spite of extensive studies. Deciphering the interactions between proinflammatory S100A9 protein and Aß42 peptide in Alzheimer's disease is fundamental since inflammation plays a central role in the disease onset. Here we use innovative charge detection mass spectrometry (CDMS) together with biophysical techniques to provide mechanistic insight into the co-aggregation process and differentiate amyloid complexes at a single particle level. Combination of mass and charge distributions of amyloids together with reconstruction of the differences between them and detailed microscopy reveals that co-aggregation involves templating of S100A9 fibrils on the surface of Aß42 amyloids. Kinetic analysis further corroborates that the surfaces available for the Aß42 secondary nucleation are diminished due to the coating by S100A9 amyloids, while the binding of S100A9 to Aß42 fibrils is validated by a microfluidic assay. We demonstrate that synergy between CDMS, microscopy, kinetic and microfluidic analyses opens new directions in interdisciplinary research.

4.
Pharm Res ; 36(5): 77, 2019 Apr 01.
Article in English | MEDLINE | ID: mdl-30937539

ABSTRACT

PURPOSE: To explore how the natural heterogeneity of human coagulation factor VIII (FVIII) and the processing of its B-domain specifically modulate protein aggregation. METHODS: Recombinant FVIII (rFVIII) molecular species containing 70% or 20% B-domain, and B-domain-deleted rFVIII (BDD-rFVIII), were separated from full-length recombinant FVIII (FL-rFVIII). Purified human plasma-derived FVIII (pdFVIII) was used as a comparator. Heterogeneity and aggregation of the various rFVIII molecular species, FL-rFVIII and pdFVIII were analysed by SDS-PAGE, dynamic light scattering, high-performance size-exclusion chromatography and flow cytometry-based particle analysis. RESULTS: FL-rFVIII and pdFVIII were heterogeneous in nature and demonstrated similar resistance to aggregation under physical stress. Differences were observed between these and among rFVIII molecular species. FVIII molecular species exhibited diverging aggregation pathways dependent on B-domain content. The propensity to form aggregates increased with decreasing proportions of B-domain, whereas the opposite was observed for oligomer formation. Development of cross-ß sheet-containing aggregates in BDD-rFVIII induced effective homologous seeding and faster aggregation. Naturally heterogeneous FL-rFVIII and pdFVIII displayed the lowest propensity to aggregate in all experiments. CONCLUSIONS: These results demonstrate that pdFVIII and FL-rFVIII have similar levels of molecular heterogeneity, and suggest that heterogeneity and the B-domain are involved in stabilising FVIII by modulating its aggregation pathway.


Subject(s)
Factor VIII/chemistry , Peptide Fragments/chemistry , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Factor VIII/isolation & purification , Humans , Mass Spectrometry , Peptide Fragments/isolation & purification , Protein Aggregates , Protein Stability , Protein Structural Elements , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
5.
Clin Immunol ; 198: 62-70, 2019 01.
Article in English | MEDLINE | ID: mdl-30389480

ABSTRACT

The mechanism of the efficacy of Intravenous immunoglobulins (IVIG) in autoimmune and inflammatory diseases is not well understood. This study aimed at understanding mechanisms of IVIG-mediated suppression of effector cell activities of peripheral blood mononuclear cells (PBMC) in antibody-dependent cellular cytotoxicity (ADCC). We were particularly interested in CD56dim NK cells, the main ADCC effector cells in PBMC. Exposure of PBMC to IVIG for at least 48 h induced a caspase-3-dependent apoptotic cell death of CD56dim NK cells without affecting CD56bright NK cells. Induction of apoptosis in CD56dim NK cells and concomitant suppression of ADCC effector activities of PBMC was associated with the monomer fraction of IVIG. Moreover, it was independent of IgG sialyation, did not depend on engagement of FcγRIII and could not be mimicked by IVIG (Fab')2 or IVIG Fc preparations. The described effect could contribute to the reduction of peripheral NK cells observed during IVIG therapy in patients.


Subject(s)
Antibody-Dependent Cell Cytotoxicity/drug effects , Apoptosis/drug effects , CD56 Antigen/analysis , Immunoglobulins, Intravenous/pharmacology , Killer Cells, Natural/drug effects , Leukocytes, Mononuclear/immunology , Humans , Killer Cells, Natural/immunology , Receptors, IgG/analysis
6.
Pharm Res ; 32(9): 2863-76, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25788448

ABSTRACT

PURPOSE: Sub-visible particles were shown to facilitate unwanted immunogenicity of protein therapeutics. To understand the root cause of this phenomenon, a comprehensive analysis of these particles is required. We aimed at establishing a flow-cytometry-based technology to analyze the amount, size distribution and nature of sub-visible particles in protein solutions. METHODS: We adjusted the settings of a BD FACS Canto II by tuning the forward scatter and the side scatter detectors and by using size calibration beads to facilitate the analysis of particles with sizes below 1 µM. We applied a combination of Bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt) and DCVJ (9-(2,2-dicyanovinyl)julolidine) to identify specific characteristics of sub-visible particles. RESULTS: The FACS technology allows the analysis of particles between 0.75 and 10 µm in size, requiring relatively small sample volumes. Protein containing particles can be distinguished from non-protein particles and cross-ß-sheet structures contained in protein particles can be identified. CONCLUSIONS: The FACS technology provides robust and reproducible results with respect to number, size distribution and specific characteristics of sub-visible particles between 0.75 and 10 µm in size. Our data for number and size distribution of particles is in good agreement with results obtained with the state-of-the-art technology micro-flow imaging.


Subject(s)
Proteins/chemistry , Solutions/chemistry , Anilino Naphthalenesulfonates/chemistry , Calibration , Flow Cytometry/methods , Particle Size , Protein Structure, Secondary
7.
Biochemistry ; 53(40): 6370-81, 2014 Oct 14.
Article in English | MEDLINE | ID: mdl-25216081

ABSTRACT

The introduction of weak, hydrophobic interactions between fluorescent protein domains (FPs) can substantially increase the dynamic range (DR) of Förster resonance energy transfer (FRET)-based sensor systems. Here we report a comprehensive thermodynamic characterization of the stability of a range of self-associating FRET pairs. A new method is introduced that allows direct quantification of the stability of weak FP interactions by monitoring intramolecular complex formation as a function of urea concentration. The commonly used S208F mutation stabilized intramolecular FP complex formation by 2.0 kCal/mol when studied in an enhanced cyan FP (ECFP)-linker-enhanced yellow FP (EYFP) fusion protein, whereas a significantly weaker interaction was observed for the homologous Cerulean/Citrine FRET pair (ΔG0(o-c) = 0.62 kCal/mol). The latter effect could be attributed to two mutations in Cerulean (Y145A and H148D) that destabilize complex formation with Citrine. Systematic analysis of the contribution of residues 125 and 127 at the dimerization interface in mOrange.linker.mCherry fusion proteins yielded a toolbox of new mOrange-mCherry combinations that allowed tuning of their intramolecular interaction from very weak (ΔG0(o-c) = .0.39 kCal/mol) to relatively stable (ΔG0(o-c) = 2.2 kCal/mol). The effects of these mutations were also studied by monitoring homodimerization of mCherry variants using fluorescence anisotropy. These mutations affected intramolecular and intermolecular domain interactions similarly, although FP interactions were found to be stronger in the latter. The knowledge thus obtained allowed successful construction of a red-shifted variant of the bile acid FRET sensor BAS-1 by replacement of the self-associating Cerulean-Citrine pair by mOrange.mCherry variants with a similar intramolecular affinity. Our findings thus allow a better understanding of the subtle but important role of intramolecular domain interactions in current FRET sensors and help guide the construction of new sensors using modular design strategies.


Subject(s)
Biosensing Techniques , Amino Acid Substitution , Bile Acids and Salts/chemistry , Fluorescence Polarization , Fluorescence Resonance Energy Transfer , Hydrophobic and Hydrophilic Interactions , Luminescent Proteins/chemistry , Protein Binding , Protein Denaturation , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Stability , Thermodynamics , Urea/chemistry , Red Fluorescent Protein
8.
Pharm Res ; 30(11): 2855-67, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23775444

ABSTRACT

PURPOSE: Human factor VIIa (FVIIa) is commonly used as bypassing therapy to treat bleeding episodes in hemophilia patients with neutralizing antibodies to factors VIII (FVIII) or IX (FIX). There is a need for a suitable animal model to assess the immunogenicity of new FVIIa products during preclinical development. The aim of this study was the design of a novel transgenic mouse model with immune tolerance to human FVIIa. METHODS: The model was generated by transgenic expression of human F7 cDNA. FVIIa-specific immune responses after treatment with human FVIIa were assessed by analyzing circulating antibodies, antibody producing plasma cells and CD4(+) T cells. RESULTS: In contrast to wild-type mice, human FVII transgenic mice did not develop antibodies when treated with human FVIIa. The immune tolerance was specific and could be broken by application of human FVIIa together with a strong stimulus of the innate immune system. Break of tolerance was associated with increased numbers of pro-inflammatory FVIIa-specific CD4(+) T cells. CONCLUSIONS: The new mouse model is suitable to study the influence of the innate immune system on maintenance and break of immune tolerance against FVIIa and could be used to assess the immunogenicity of new FVIIa products during pre-clinical development.


Subject(s)
Factor VIIa/genetics , Factor VIIa/immunology , Immune Tolerance , Transgenes , Animals , CD8-Positive T-Lymphocytes/immunology , DNA, Complementary/genetics , Factor VIIa/therapeutic use , Female , Humans , Immunity, Innate , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Animal
9.
Chem Commun (Camb) ; 47(33): 9333-5, 2011 Sep 07.
Article in English | MEDLINE | ID: mdl-21766106

ABSTRACT

The transfer of the cooperative self-assembled fibrils to a gold substrate has been studied by means of scanning probe microscopy techniques revealing the crucial role of the early formation of a monolayer.

10.
J Mol Biol ; 396(1): 60-74, 2010 Feb 12.
Article in English | MEDLINE | ID: mdl-19913026

ABSTRACT

Amyloid formation is a universal behavior of proteins central to many important human pathologies and industrial processes. The extreme stability of amyloids towards chemical and proteolytic degradation is an acquired property compared to the precursor proteins and is a major prerequisite for their accumulation. Here, we report a study on the lability of human insulin amyloid as a function of pH and amyloid ageing. Using a range of methods such as atomic force microscopy, thioflavin T fluorescence, circular dichroism, and gas-phase electrophoretic mobility macromolecule analysis, we probed the propensity of human insulin amyloid to propagate or dissociate in a wide span of pH values and ageing in a low concentration regime. We generated a three-dimensional amyloid lability landscape in coordinates of pH and amyloid ageing, which displays three distinctive features: (i) a maximum propensity to grow near pH 3.8 and an age corresponding to the inflection point of the growth phase, (ii) an abrupt cutoff between growth and disaggregation at pH 8-10, and (iii) isoclines shifted towards older age during the amyloid growth phase at pH 4-9, reflecting the greater stability of aged amyloid. Thus, lability of amyloid strongly depends on the ionization state of insulin and on the structure and maturity of amyloid fibrils. The stability of insulin amyloid towards protease K was assessed by using real-time atomic force microscopy and thioflavin T fluorescence. We estimated that amyloid fibrils can be digested both from the free ends and within the length of the fibril with a rate of ca 4 nm/min. Our results highlight that amyloid structures, depending on solution conditions, can be less stable than commonly perceived. These results have wide implications for understanding the propagation of amyloids via a seeding mechanism as well as for understanding their natural clearance and dissociation under solution conditions unfavorable for amyloid formation in biological systems and industrial applications.


Subject(s)
Amyloid/chemistry , Amyloid/metabolism , Endopeptidase K/metabolism , Insulin/chemistry , Insulin/metabolism , Models, Molecular , Amyloid/ultrastructure , Benzothiazoles , Circular Dichroism , Electrophoresis , Enzyme Stability , Humans , Hydrogen-Ion Concentration , Kinetics , Microscopy, Atomic Force , Protein Binding , Protein Structure, Quaternary , Solutions , Spectrum Analysis , Thiazoles/metabolism , Time Factors
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