Monoclonal antibodies against tumor necrosis factor-alpha (TNFα) are widely used for treatment of inflammatory diseases. However, despite the inhibitory effect this class of drugs has on the immune... Show moreMonoclonal antibodies against tumor necrosis factor-alpha (TNFα) are widely used for treatment of inflammatory diseases. However, despite the inhibitory effect this class of drugs has on the immune system, anti-drug antibodies are often formed with continuous use. Particles formed during stress conditions, which can be used to simulate storage and handling conditions of commercial antibodies, have previously been associated with the formation of anti-drug antibodies. This study investigates the relationship between particles, oligomerization, folding and chemical degradation on the in vitro cytokine response toward the TNFα inhibitor adalimumab. Adalimumab aggregates generated using stir and heat stress were fractionated into distinct sub-populations, and their structure and immunogenic potential were evaluated. A chemically degraded sample of adalimumab was included to compare particle composition with the milder accelerated heat and stir stressed conditions. Particles from stressed adalimumab samples induced elevated cytokine levels and CD4+ T cell proliferation in vitro compared to non-stressed samples. Samples enriched with both submicron and subvisible particles of adalimumab induced the strongest cytokine release and the strongest CD4+ T cell proliferation despite maintaining some TNFα inhibitory functionality. Samples that were stressed and subsequently purified of subvisible and submicron particles did not elicit a significantly higher cytokine response or show increased CD4+ T cell proliferation compared to a non-stressed sample. Oxidation-induced chemical modifications in adalimumab, mainly in Met, His, Trp, and Tyr, were not found to be sufficient in absence of particle formation to induce increased CD4+ T cell proliferation or cytokine release despite less decreased TNFα inhibitory activity of adalimumab. These observations provide further evidence that particles do indeed potentiate the immunogenic potential of adalimumab. Show less
Heydarian, M.; Oak, P.; Zhang, X.; Kamgari, N.; Kindt, A.S.D.; Koschlig, M.; ... ; Hilgendorff, A. 2022
Introduction Chronic lung disease, that is, bronchopulmonary dysplasia (BPD) is the most common complication in preterm infants and develops as a consequence of the misguided formation of the gas... Show moreIntroduction Chronic lung disease, that is, bronchopulmonary dysplasia (BPD) is the most common complication in preterm infants and develops as a consequence of the misguided formation of the gas-exchange area undergoing prenatal and postnatal injury. Subsequent vascular disease and its progression into pulmonary arterial hypertension critically determines long-term outcome in the BPD infant but lacks identification of early, disease-defining changes. Methods We link impaired bone morphogenetic protein (BMP) signalling to the earliest onset of vascular pathology in the human preterm lung and delineate the specific effects of the most prevalent prenatal and postnatal clinical risk factors for lung injury mimicking clinically relevant conditions in a multilayered animal model using wild-type and transgenic neonatal mice. Results We demonstrate (1) the significant reduction in BMP receptor 2 (BMPR2) expression at the onset of vascular pathology in the lung of preterm infants, later mirrored by reduced plasma BMP protein levels in infants with developing BPD, (2) the rapid impairment (and persistent change) of BMPR2 signalling on postnatal exposure to hyperoxia and mechanical ventilation, aggravated by prenatal cigarette smoke in a preclinical mouse model and (3) a link to defective alveolar septation and matrix remodelling through platelet derived growth factor-receptor alpha deficiency. In a treatment approach, we partially reversed vascular pathology by BMPR2-targeted treatment with FK506 in vitro and in vivo. Conclusion We identified impaired BMP signalling as a hallmark of early vascular disease in the injured neonatal lung while outlining its promising potential as a future biomarker or therapeutic target in this growing, high-risk patient population. Show less
Nanoparticle tracking analysis (NTA) has attracted great interest for application in the field of submicron particle characterization for biopharmaceuticals. It has the virtue of direct sample... Show moreNanoparticle tracking analysis (NTA) has attracted great interest for application in the field of submicron particle characterization for biopharmaceuticals. It has the virtue of direct sample visualization and particle-by-particle tracking, but the complexity of method development has limited its routine applicability. We systematically evaluated data collection and processing parameters as well as sample handling methods using shake-stressed protein samples. The camera shutter and gain were identified as the key factors influencing NTA results. We also demonstrated that sample filtration was necessary for NTA analysis if there were high numbers of micron particles, whereas the choice of filter membrane was critical for data quality. Sample dilution into corresponding formulation buffer did not affect particle size distributions in our study. Finally, NTA analysis exhibited excellent repeatability in intraday comparison of multiple measurements on the same sample and interday comparison on different batches of samples. Shaking-induced protein aggregation could also be sensitively monitored by NTA. In conclusion, NTA analysis can be used as a robust stability-indicating method for the characterization of proteinaceous submicron particles and thereby complement other analytical methods, provided that consistent sample handling and parametric settings are established for the specific case study. Show less
