RESUMO
Protein post-translational modification (PTM) plays an important role in many biological processes; of which glycosylation is arguably one of the most complex and diverse modifications and is crucial for the safety and efficacy of biotherapeutic proteins. Mass spectrometric characterization of protein glycosylation is well established with clear advantages and disadvantages; on one hand it is precise and information-rich, as well as being relative inexpensive in terms of the reagents and consumables despite the instrumentation cost and, depending on the method, can give site specific information; on the other hand it generally suffers from low throughput, restriction to largely purified samples and is less quantitative, especially for sialylated glycan species. Here, we describe a high throughput, site-specific, targeted mass spectrometric peptide mapping approach to quickly screen/rank candidate production cell lines and culture conditions that give favourable glycosylation profiles directly from conditioned culture media for an Fc-fusion protein. The methodology is fully compatible with automation and combines the speed of 'top-down' mass spectrometry with the site-specific information of 'bottom-up' mass spectrometry. In addition, this strategy can be used for multi-attribute product quality screening/monitoring as an integral part of cell line selection and process development.
RESUMO
In response to infections and irritants, the respiratory epithelium releases the alarmin interleukin (IL)-33 to elicit a rapid immune response. However, little is known about the regulation of IL-33 following its release. Here we report that the biological activity of IL-33 at its receptor ST2 is rapidly terminated in the extracellular environment by the formation of two disulphide bridges, resulting in an extensive conformational change that disrupts the ST2 binding site. Both reduced (active) and disulphide bonded (inactive) forms of IL-33 can be detected in lung lavage samples from mice challenged with Alternaria extract and in sputum from patients with moderate-severe asthma. We propose that this mechanism for the rapid inactivation of secreted IL-33 constitutes a 'molecular clock' that limits the range and duration of ST2-dependent immunological responses to airway stimuli. Other IL-1 family members are also susceptible to cysteine oxidation changes that could regulate their activity and systemic exposure through a similar mechanism.