A host-cell-related proteolytic activity was identified in a recombinant Fc-fusion protein production process. This report describes the strategy applied to characterize and isolate the enzyme ...responsible for this degradation by combining cell culture investigation and dedicated analytical tools. After isolation and sequencing of the clipped fragment generated in post-capture material, enzymatic activity was traced in different culture conditions, allowing identification of viable CHO cells as the source of protease. Inhibitors and pH screenings showed that the enzyme belongs to an aspartic protease family and is preferably active at acidic pH. The protease was isolated by purification on a pepstatin A column and characterized as a protein related to cathepsin D. An additional metallo-protease inhibited by EDTA was identified with an optimum activity at neutral pH. This study is an example of how quality and stability of therapeutic recombinant molecules are strongly influenced by cell culture parameters. Biotechnol. Bioeng. 2009; 104: 1132-1141.
Permafrost in the NE European Russian Arctic is suffering from some of the highest degradation rates in the world. The rising mean annual air temperature causes warming permafrost, the increase in ...the active layer thickness (ALT), and the reduction of the permafrost extent. These phenomena represent a serious risk for infrastructures and human activities. ALT characterization is important to estimate the degree of permafrost degradation. We used a multidisciplinary approach to investigate the ALT distribution in the Khanovey railway station area (close to Vorkuta, Arctic Russia), where thaw subsidence leads to railroad vertical deformations up to 2.5 cm/year. Geocryological surveys, including vegetation analysis and underground temperature measurements, together with the faster and less invasive electrical resistivity tomography (ERT) geophysical method, were used to investigate the frozen/unfrozen ground settings between the railroad and the Vorkuta River. Borehole stratigraphy and landscape microzonation indicated a massive prevalence of clay and silty clay sediments at shallow depths in this area. The complex refractive index method (CRIM) was used to integrate and quantitatively validate the results. The data analysis showed landscape heterogeneity and maximum ALT and permafrost thickness values of about 7 and 50 m, respectively. The active layer was characterized by resistivity values ranging from about 30 to 100 Ωm, whereas the underlying permafrost resistivity exceeded 200 Ωm, up to a maximum of about 10 kΩm. In the active layer, there was a coexistence of frozen and unfrozen unconsolidated sediments, where the ice content estimated using the CRIM ranged from about 0.3 – 0.4 to 0.9. Moreover, the transition zone between the active layer base and the permafrost table, whose resistivity values ranged from 100 to 200 Ωm for this kind of sediments, showed ice contents ranging from 0.9 to 1.0. Taliks were present in some depressions of the study area, characterized by minimum resistivity values lower than 10 Ωm. This thermokarst activity was more active close to the railroad because of the absence of insulating vegetation. This study contributes to better understanding of the spatial variability of cryological conditions, and the result is helpful in addressing engineering solutions for the stability of the railway.
N-glycosylation plays a key role in modulating the bioactivity of monoclonal antibodies (mAbs), as well as the light chain (LC) isotype can influence their physicochemical properties. However, ...investigating the impact of such features on mAbs conformational behavior is a big challenge, due to the very high flexibility of these biomolecules. In this work we investigate, by accelerated molecular dynamics (aMD), the conformational behavior of two commercial immunoglobulins G1 (IgG1), representative of κ and λ LCs antibodies, in both their fucosylated and afucosylated forms. Our results show, through the identification of a stable conformation, how the combination of fucosylation and LC isotype modulates the hinge behavior, the Fc conformation and the position of the glycan chains, all factors potentially affecting the binding to the FcγRs. This work also represents a technological enhancement in the conformational exploration of mAbs, making aMD a suitable approach to clarify experimental results.
Although fractures had high mortality and morbidity, many studies proved that fracture risk might be decreased by pharmacological therapy, although a low treatment adherence rate is observed. The aim ...of this study was to identify factors associated with osteoporosis treatment in postmenopausal women.A cross-sectional study was carried out from March to August 2013 at the primary care setting. Postmenopausal women were recruited. A standardized questionnaire was applied. Women who were using at least one of the following drugs at the moment of the survey were considered as current treatment: bisphosphonates, raloxifene, estrogen, calcitonin, teriparatide, or strontium ranelate. Women who had used any of the mentioned medications before the study were considered as past treatment.Of the 1025 women included in the study, 8% were on current treatment, 5.7% had past treatment, and 86.3% had not received treatment. Treated women (either current or past) had a higher rate of osteoarthritis, had more falls, had higher education level, presented a higher rate of private health insurance, and received more information about osteoporosis. They also had more dual-energy x-ray absorptiometry (DXA) scans and were more frequently diagnosed with osteoporosis by these DXA scans. The factors independently associated with treatment in the regression analysis were the DXA scan itself, the diagnosis of osteoporosis by DXA, and information about osteoporosis.Current and past treatments of osteoporosis were associated with DXA and information. These results suggest that some measures to inform women about osteoporosis and or even the popularization of DXA scans could improve the treatment.
The healthcare industry is continuously evolving with innovative discoveries and therapies, and at the same time, there is a decline in the research and development productivity leading to an ...increased cost for payers, providers, and patients. Despite the benefits that digital technologies can have on healthcare innovation, such a highly regulated industry often relies on proven-established technologies and organizational procedures that can be at odds with the new logics of digital innovation. In this article, we will be introducing a digital innovation management (DIM) framework that guides the pursuit of digital innovations in a phase-appropriate and incremental setup (e.g., scale, costs, risks, value evaluations, policies, and resources). The framework is designed and validated through an iterative process of continuous adaptation with local practices in a biopharmaceutical company. DIM provides practical guidance to drive digital innovations that entail different logics compared to traditional innovations, by improving the visibility of the digital innovation process and increasing organizational confidence in pursuing digital innovations and enhancing decision-making effectiveness.
Digital transformation (DT) is a critical area of focus as it disruptively transforms organizational business models, processes, and products. Despite being a prominent topic among information system ...scholars, the research has mostly centered around technological aspects, and there is a need to broaden the research approach to increase the understanding of DT's intricate dynamics, interdisciplinary nature, and cross-level perspectives, with practical guidance, while providing actionable insights for organizations. Through action design research, we elaborate a practical Digital-by-Design (DbD) framework grounded in theoretical foundations, to emphasize the interconnected dimensions of people, process and technology for enhancing DT feasibility and viability. This research contributes to a deeper understanding of DT initiatives by recognizing the significance of: 1) diverse organizational, process, and industry needs; 2) individual uniqueness concerning DT; 3) technology integration and scale-up patterns; 4) cross-level empirical perspectives; and 5) an interdisciplinary approach by supporting organizations when navigating DT complexities and dynamics. Recognizing the practical managerial challenges, the study offers a comprehensive guidance for designing and implementing a DT into the business processes.
Currently, monoclonal antibodies (mAbs) are the most used biopharmaceuticals for human therapy. One of the key aspects in their development is the control of effector functions mediated by the ...interaction between fragment crystallizable (Fc) and Fcγ receptors, which is a secondary mechanism of the action of biotherapeutics. N-glycosylation at the Fc portion can regulate these mechanisms, and much experimental evidence suggests that modifications of glycosidic chains can affect antibody binding to FcγRIIIa, consequently impacting the immune response. In this work, we try to elucidate via in silico procedures the structural role exhibited by glycans, particularly fucose, in mAb conformational freedom that can potentially affect the receptor recognition. By using adalimumab, a marketed IgG1, as a general template, after rebuilding its three-dimensional (3D) structure through homology modeling approaches, we carried out molecular dynamics simulations of three differently glycosylated species: aglycosylated, afucosylated, and fucosylated antibody. Trajectory analysis showed different dynamical behaviors and pointed out that sugars can influence the overall 3D structure of the antibody. As a result, we propose a putative structural mechanism by which the presence of fucose introduces conformational constraints in the whole antibody and not only in the Fc domain, preventing a conformation suitable for the interaction with the receptor. As secondary evidence, we observed a high flexibility of the antibodies that is translated into an asymmetric behavior of Fab portions shown by all the simulated biopolymers, making the dynamical asymmetry a new, to our knowledge, molecular aspect that may be further investigated. In conclusion, these findings can help understand the contribution of sugars on the structural architecture of mAbs, paving the way to novel strategies of pharmaceutical development.
There is an increasing interest in the generation of Fc‐fusion molecules to exploit the effector functions of Fc and the fusion partner, towards improving the therapeutic potential. The Fc‐fusion ...molecules have unique structural and functional attributes that impart various advantages. However, the manufacturing of Fc‐fusion molecules possesses certain challenges in the biopharmaceutical development. The fusion of unnaturally occurring two or more domains in a construct can pose problems for proper folding and are prone to aggregation and degradation. Reshuffling of disulfide bridges represents a posttranslational event that affects folding. This can play a critical role in the correct structure of a molecule and leads to structural heterogeneity in biotherapeutics; it may also impact the in vivo biological activities, safety, and efficacy of the biopharmaceutical. Our work presents an investigation case of a doublet band, as observed only in nonreducing sodium dodecyl sulfate ‐ polyacrylamide gel electrophoresis (SDS‐PAGE) for a bi‐specific, N‐ and C‐terminal Fc‐fusion molecule. Other characterization and orthogonal methods from the analytical panel did not indicate the presence of two distinct species, including the orthogonal CE‐SDS (Caliper Lab Chip GXII). Therefore, it was necessary to determine if the phenomenon was an analytical artifact or a real variant of our Fc‐fusion molecule. With the comprehensive mass spectrometry‐based characterization, we were able to determine that the doublet band was related to the reshuffling of one disulfide bridge in one of the fused domains. Our work illustrates the application of nonreducing peptide mapping by mass spectrometry to characterize and identify disulfide variants in a complex N‐ and C‐terminal Fc‐fusion molecule, and further adoption to monitor the disulfide structural variants in the intermediate process samples to drive the manufacturing of a consistent product with the desired quality attributes.
A hyperinflammatory syndrome (HIS) may cause a life-threatening acute respiratory distress syndrome (ARDS) in patients with COVID-19 pneumonia.
A prospective series of 100 consecutive patients ...admitted to the Spedali Civili University Hospital in Brescia (Italy) between March 9th and March 20th with confirmed COVID-19 pneumonia and ARDS requiring ventilatory support was analyzed to determine whether intravenous administration of tocilizumab (TCZ), a monoclonal antibody that targets the interleukin 6 (IL-6) receptor, was associated with improved outcome. Tocilizumab was administered at a dosage of 8 mg/kg by two consecutive intravenous infusions 12 h apart. A third infusion was optional based on clinical response.
The outcome measure was an improvement in acute respiratory failure assessed by means of the Brescia COVID Respiratory Severity Score (BCRSS 0 to 8, with higher scores indicating higher severity) at 24–72 h and 10 days after tocilizumab administration.
Out of 100 treated patients (88 M, 12 F; median age: 62 years), 43 received TCZ in the intensive care unit (ICU), while 57 in the general ward as no ICU beds were available. Of these 57 patients, 37 (65%) improved and suspended noninvasive ventilation (NIV) (median BCRSS: 1 IQR 0–2), 7 (12%) patients remained stable in NIV, and 13 (23%) patients worsened (10 died, 3 were admitted to ICU). Of the 43 patients treated in the ICU, 32 (74%) improved (17 of them were taken off the ventilator and were discharged to the ward), 1 (2%) remained stable (BCRSS: 5) and 10 (24%) died (all of them had BCRSS≥7 before TCZ). Overall at 10 days, the respiratory condition was improved or stabilized in 77 (77%) patients, of whom 61 showed a significant clearing of diffuse bilateral opacities on chest x-ray and 15 were discharged from the hospital. Respiratory condition worsened in 23 (23%) patients, of whom 20 (20%) died.
All the patients presented with lymphopenia and high levels of C-reactive protein (CRP), fibrinogen, ferritin and IL-6 indicating a HIS. During the 10-day follow-up, three cases of severe adverse events were recorded: two patients developed septic shock and died, one had gastrointestinal perforation requiring urgent surgery and was alive at day 10.
In conclusion, our series showed that COVID-19 pneumonia with ARDS was characterized by HIS. The response to TCZ was rapid, sustained, and associated with significant clinical improvement.
In this work, a theoretical-computational method is applied to study the deamidation reaction, a critical post-translational modification in proteins, using a simple model molecule in solution. The ...method allows one to comprehensively address the environmental effect, thereby enabling one to accurately derive the kinetic rate constants for the three main steps of the deamidation process. The results presented, in rather good agreement with the available experimental data, underline the necessity for a rigorous treatment of environmental factors and a precise kinetic model to correctly assess the overall kinetics of the deamidation reaction.
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