Tracking the Behavior of Monoclonal Antibody Product Quality Attributes Using a Multi-Attribute Method Workflow.

The multi-attribute method (MAM) is a liquid chromatography-mass spectrometry based method that is used to directly characterize and monitor many product quality attributes and impurities on biotherapeutics, most commonly at the peptide level. It utilizes high-resolution accurate mass spectral data which are analyzed in an automated fashion. MAM is a promising approach that is intended to replace or supplement several conventional assays with a single LC-MS analysis and can be implemented in a Current Good Manufacturing Practice environment. MAM provides accurate site-specific quantitation information on targeted attributes and the nontargeted new peak detection function allows to detect new peaks as impurities, modifications, or sequence variants when comparing to a reference sample. The high resolution MAM workflow was applied here for three independent case studies. First, to monitor the behavior of monoclonal antibody product quality attributes over the course of a 12-day cell culture experiment providing an insight into the behavior and dynamics of product attributes throughout the process. Second, the workflow was applied to test the purity and identity of a product through analysis of samples spiked with host cell proteins. Third, through the comparison of a drug product and a biosimilar with known sequence variants. The three case studies presented here, clearly demonstrate the robustness and accuracy of the MAM workflow that implies suitability for deployment in the regulated environment.

Computational analysis and enzyme assay of inhibitor response to disease single nucleotide polymorphisms (SNPs) in lipoprotein lipase.

Lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of the triglyceride (TG) core of circulating TG-rich lipoproteins, chylomicrons, and very low-density lipoproteins. The enzyme has been established as an efficacious and safe therapeutic target for the management of obesity. Here, a systematic profile of the lipase inhibitor response of three anti-obesity agents (Orlistat, Lipstatin, and Cetilistat) to clinical LPL missense mutations arising from disease single nucleotide polymorphisms (SNPs) was established by integrating complex structure modeling, virtual mutagenesis, molecular dynamics (MD) simulations, binding energy analysis, and radiolabeled TG hydrolysis assays. The profile was then used to characterize the resistance and sensitivity of systematic mutation-inhibitor pairs. It is suggested that the Orlistat and Lipstatin have a similar response profile to the investigated mutations due to their homologous chemical structures, but exhibit a distinct profile to that of Cetilistat. Most mutations were predicted to have a modest or moderate effect on inhibitor binding; they are located far away from the enzyme active site and thus can only influence the binding limitedly. A number of mutations were found to sensitize or cause resistance for lipase inhibitors by directly interacting with the inhibitor ligands or by indirectly addressing allosteric effect on enzyme active site. Long-term MD simulations revealed a different noncovalent interaction network at the complex interfaces of Orlistat with wild-type LPL as well as its sensitized mutant H163R and resistant mutant I221T.

MicroRNA-410 regulated lipoprotein lipase variant rs13702 is associated with stroke incidence and modulated by diet in the randomized controlled PREDIMED trial.

BACKGROUND: MicroRNAs have emerged as important epigenetic regulators in cardiovascular diseases (CVDs). Using an observational meta-analysis design, we previously characterized a gain-of-function microRNA-410 target site polymorphism (rs13702T>C) in the 3'untranslated region of the lipoprotein lipase (LPL) gene. The C allele was associated with lower triglycerides, and this association was modulated by fat intake. OBJECTIVES: We aimed to extend our findings by assessing the interaction between the rs13702 polymorphism and fat intake on triglycerides at baseline and longitudinally by using a dietary intervention design. We also examined as a primary outcome the association of this variant with CVD incidence and its modulation by the Mediterranean diet (MedDiet). DESIGN: We studied 7187 participants in the PREDIMED (Prevención con Dieta Mediterránea) randomized trial that tested a MedDiet intervention compared with a control diet, with a median 4.8-y follow-up. LPL polymorphisms and triglycerides were determined and CVD assessed. Gene-diet interactions for triglycerides were analyzed at baseline (n = 6880) and after a 3-y intervention (n = 4131). Oxidative stress parameters were investigated in a subsample. RESULTS: The rs13702T>C polymorphism was strongly associated with lower triglycerides in C allele carriers and interacted synergistically with dietary monounsaturated (P = 0.038) and unsaturated fat intake (P = 0.037), decreasing triglycerides at baseline. By 3 y, we observed a gene-diet interaction (P = 0.025) in which the C allele was associated with a greater reduction in triglycerides after intervention with MedDiet, high in unsaturated fat. Although the polymorphism was associated with lower stroke risk (HR: 0.74; 95% CI: 0.57, 0.97; P = 0.029 per C allele), this association reached statistical significance only in the MedDiet intervention (HR: 0.58; 95% CI: 0.37, 0.91; P = 0.019 in C compared with TT carriers), not in the control group (HR: 0.94; 95% CI: 0.55, 1.59; P = 0.805). CONCLUSION: We report a novel association between a microRNA target site variant and stroke incidence, which is modulated by diet in terms of decreasing triglycerides and possibly stroke risk in rs13702 C allele carriers after a high-unsaturated fat MedDiet intervention.

Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase.

Mobilization of fatty acids from triglyceride stores in adipose tissue requires lipolytic enzymes. Dysfunctional lipolysis affects energy homeostasis and may contribute to the pathogenesis of obesity and insulin resistance. Until now, hormone-sensitive lipase (HSL) was the only enzyme known to hydrolyze triglycerides in mammalian adipose tissue. Here, we report that a second enzyme, adipose triglyceride lipase (ATGL), catalyzes the initial step in triglyceride hydrolysis. It is interesting that ATGL contains a "patatin domain" common to plant acyl-hydrolases. ATGL is highly expressed in adipose tissue of mice and humans. It exhibits high substrate specificity for triacylglycerol and is associated with lipid droplets. Inhibition of ATGL markedly decreases total adipose acyl-hydrolase activity. Thus, ATGL and HSL coordinately catabolize stored triglycerides in adipose tissue of mammals.

Maturation of lipoprotein lipase in the endoplasmic reticulum. Concurrent formation of functional dimers and inactive aggregates.

The maturation of lipoprotein lipase (LPL) into a catalytically active enzyme was believed to occur only after its transport from the endoplasmic reticulum (ER) to the Golgi apparatus. To test this hypothesis, LPL located in these two subcellular compartments was separated and compared. Heparin affinity chromatography resolved low affinity, inactive LPL displaying ER characteristics from a high affinity, active fraction exhibiting both ER and Golgi forms. The latter forms were further separated by beta-ricin chromatography and were found to have comparable activities per unit of LPL mass. Thus, LPL must reach a functional conformation in the ER. Active LPL, regardless of its cellular location, exhibited the expected dimer conformation. However, inactive LPL, found only in the ER, was highly aggregated. Kinetic analysis indicated a concurrent formation of LPL dimer and aggregate and indicated that the two forms have dissimilar fates. Whereas the dimer remained stable even when confined to the ER, the aggregate was degraded. Degradation rates were not affected by proteasomal or lysosomal inhibitors but were markedly reduced by ATP depletion. Lowering the redox potential in the ER by dithiothreitol caused the dimer to associate with calnexin, BiP, and protein-disulfide isomerase to form large, inactive complexes; dithiothreitol removal induced complex dissociation with restoration of the functional LPL dimer. In contrast, the LPL aggregate was only poorly associated with ER chaperones, appearing to be trapped in an irreversible, inactive conformation destined for ER degradation.

Lipoprotein lipase: role of intramolecular disulfide bonds in enzyme catalysis.

Lipoprotein lipase (LPL) catalyzes the hydrolysis of the triacylglycerol component of triacylglycerol-rich lipoproteins. There are 4 cysteine pairs that are completely conserved among LPLs of all species known. We examined the functional importance of each of the cysteine pairs in enzyme catalysis by examining LPLs produced in Cos cells by transfection. Immunoreactive LPL was produced by vectors encoding the wildtype LPL and each of the 4 cysteine-pair mutant LPLs. Enzyme activity was detectable in the wildtype enzyme, but not in 3 of the 4 Cys-->Ser mutant enzymes (C216S/C239S, C264S/C275S, and C278S/C283S). Interestingly, LPL activity was also present in the mutant (C418S/C438S), which affects the C-terminal cysteine pair, with a specific activity approximately 50% higher than that of wildtype. There is evidence that LPL contains two distinct domains consisting of the N-terminal three-quarters of the sequence connected by a flexible region to the C-terminal domain comprising the rest of the molecule. The conservation of catalytic function despite the disruption of the only disulfide bridge in the C-terminal domain of LPL indicates that the two domains can function independently of each other in enzyme catalysis.

Metabolism of different adipose tissues in vivo in the rat.

To explore regional differences in triglyceride retention in white adipose tissues of growing male rats, the mass of adipocytes from epididymal, retroperitoneal, inguinal, and mesenteric tissues were followed with time. In order to attempt to explain regional differences, adipose tissue metabolism was studied in vivo and in vitro. (U-14C) oleic acid in sesame oil was given by gastric gavage to conscious male and female rats, and accumulation and half-life of radioactivity measured. Lipoprotein lipase activity and lipolysis were studied in vitro. Adipocyte triglyceride mass increased linearly in all the depots during 4 months of observation.The increase in mass was more pronounced in retroperitoneal (0.31 microg) and epididymal (0.30 microg) than in mesenteric (0.11 microg) or inguinal (0.05 microg) adipocytes. In the fed state label from (U-14C) oleic acid first increased with time in liver, muscle, and adipose tissues. In the liver radioactivity peaked at 4 hours, and was not measurable in either liver or muscle after a time point between 24 hours to 1 week. In contrast label continued to increase in adipose tissues up to about 16 hours to 24 hours, suggesting transfer of label by recirculation from liver and muscle to adipose tissues. Thereafter the radioactivity decreased. When expressed per adipocyte uptake of label was not significantly different between white adipose tissues. The rate of decrease between 7 days and 4 months was, however, more rapid in mesenteric and inguinal than, particularly, epididymal, and, probably, retroperitoneal adipocytes. These results were partly parallel to in vitro data on lipoprotein lipase activity, which was not different between depots, and the rate of lipolysis, which was higher in mesenteric than other adipocytes. These results suggest that differences in weight increase of adipose tissue regions are due mainly to differences in the rate of mobilization of adipocyte triglycerides. When expressed per gram triglyceride, uptake and mobilization of label were clearly more rapid in mesenteric than other white adipose tissues. This is probably explained by a combination of a higher adipocyte density plus the metabolic characteristics of adipocytes in this depot. Since mesenteric adipose tissue is smaller than the other depots studied, the absolute contribution of this tissue to the energy supply of the body is probably not different from that of other adipose tissues, however. A large uptake and short half life was observed in interscapular adipose tissue. This region contains brown adipocytes, and the results therefore suggest that lipid uptake for thermogenic purposes is of a considerable magnitude. It was concluded that among white adipose tissues, the mesenteric tissue has a rapid turnover of triglyceride. This is probably due to a combination of a high density and specific metabolic characteristics of these adipocytes. Factors in the microenvironment of adipocytes probably contribute to the high turnover either directly, or by modification of cellular characteristics.