Pathogenic factors associated with chronic obstructive pulmonary disease (COPD), such as cigarette smoke, proinflammatory cytokines, and bacterial infections, can individually induce respiratory mucins in vitro and in vivo. Since co-presence of these factors is common in lungs of patients with COPD, we hypothesized that cigarette smoke can amplify mucin induction by bacterial exoproducts and proinflammatory cytokines, resulting in mucin hyperproduction. We demonstrated that cigarette smoke extract (CSE) synergistically increased gene expression and protein production of MUC5AC mucin induced by LPS or TNF-alpha in human airway epithelial NCI-H292 cells. CSE also enhanced expression and production of MUC5AC mucin induced by epidermal growth factor receptor (EGFR) ligands TGF-alpha and amphiregulin, as well as LPS- and TNF-alpha- induced expression and/or release of TGF-alpha and amphiregulin. Furthermore, (4-[(3-bromophenyl)amino]-6,7-diaminoquinazoline), a potent inhibitor of EGFR, blocked synergistic induction of MUC5AC mucin. H(2)O(2) mimicked the synergistic effects of CSE, while antioxidant N-acetyl-L-cysteine prevented synergistic induction of MUC5AC mucin by CSE. In a rat model of LPS-induced airway inflammation, concurrent cigarette smoke inhalation enhanced mucin content of the bronchoalveolar lavage fluid, muc5AC gene expression, and mucous cell metaplasia in the airways. These results suggest that cigarette smoke has the potential to synergistically amplify induction of respiratory mucins by proinflammatory stimuli relevant to COPD pathogenesis and contribute to mucin hyperproduction observed in patients with COPD.

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Background: Escherichia coli (E. coli) is a promising host for production of recombinant proteins (including antibodies and antibody fragments) that don’t require complex post-translational modifications such as glycosylation. During manufacturing-scale production of a one-armed antibody in E. coli (periplasmic production), variability in the degree of reduction of the antibody’s disulfide bonds was observed. This resulted in variability in the free thiol content, a potential critical product quality attribute. This work was initiated to understand and prevent the variability in the total free thiol content during manufacturing. Results: In this study, we found that the reduction in antibody’s disulfide bonds was observed to occur during homogenization and the ensuing homogenate hold step where in the antibody is exposed to redox enzymes and small molecule reductants present in homogenate. Variability in the downstream processing time between the start of homogenization and end of the homogenate hold step resulted in variability in the degree of antibody disulfide bond reduction & free thiol content. The disulfide bond reduction in the homogenate is catalyzed by the enzyme disulfide bond isomerase C (DsbC) and is highly site-specific and occurred predominantly in the intra-chain disulfide bonds present in the Fc CH2 region. Our results also imply that lack of glycans in E. coli produced antibodies may facilitate DsbC accessibility to the disulfide bond in the Fc CH2 region, resulting in its reduction. Conclusions: During E. coli antibody manufacturing processes, downstream processing steps such as homogenization and subsequent processing of the homogenate can impact degree of disulfide bond reduction in the antibody and consequently product quality attributes such as total free thiol content. Duration of the homogenate hold step should be minimized as much as possible to prevent disulfide bond reduction & free thiol formation. Other approaches such as reducing homogenate temperature, adding flocculants prior to homogenization, using enzyme inhibitors, or modulating redox environments in the homogenate should be considered to prevent antibody disulfide bond reduction during homogenization and homogenate processing steps in E. coli antibody manufacturing processes.

Background Escherichia coli (E. coli) is a promising host for production of recombinant proteins (including antibodies and antibody fragments) that don’t require complex post-translational modifications such as glycosylation. During manufacturing-scale production of a one-armed antibody in E. coli (periplasmic production), variability in the degree of reduction of the antibody’s disulfide bonds was observed. This resulted in variability in the free thiol content, a potential critical product quality attribute. This work was initiated to understand and prevent the variability in the total free thiol content during manufacturing. Results In this study, we found that the reduction in antibody’s disulfide bonds was observed to occur during homogenization and the ensuing homogenate hold step where in the antibody is exposed to redox enzymes and small molecule reductants present in homogenate. Variability in the downstream processing time between the start of homogenization and end of the homogenate hold step resulted in variability in the degree of antibody disulfide bond reduction and free thiol content. The disulfide bond reduction in the homogenate is catalyzed by the enzyme disulfide bond isomerase C (DsbC) and is highly site-specific and occurred predominantly in the intra-chain disulfide bonds present in the Fc CH2 region. Our results also imply that lack of glycans in E. coli produced antibodies may facilitate DsbC accessibility to the disulfide bond in the Fc CH2 region, resulting in its reduction. Conclusions During E. coli antibody manufacturing processes, downstream processing steps such as homogenization and subsequent processing of the homogenate can impact degree of disulfide bond reduction in the antibody and consequently product quality attributes such as total free thiol content. Duration of the homogenate hold step should be minimized as much as possible to prevent disulfide bond reduction and free thiol formation. Other approaches such as reducing homogenate temperature, adding flocculants prior to homogenization, using enzyme inhibitors, or modulating redox environments in the homogenate should be considered to prevent antibody disulfide bond reduction during homogenization and homogenate processing steps in E. coli antibody manufacturing processes.