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.

Feeding a concentrate rich in rapeseed oil improves fatty acid composition and flavor in Norwegian goat milk.

Impaired quality due to a high content of free fatty acids (FFA) and off-flavors has caused challenges in the development of Norwegian goat milk products. The present study aimed to examine the effect of lipid-supplemented concentrates on milk fat content, fatty acid composition, FFA, lipoprotein lipase activity, sensory properties, and size of milk fat globules of goat milk. Thirty goats assigned to 3 experimental groups were fed different concentrates from 60 d in milk (DIM) until late lactation (230 DIM). The diets were (1) control concentrate (no added fat); (2) control concentrate with 8% (added on air-dry basis) hydrogenated palm oil enriched with palmitic acid (POFA); and (3) control concentrate with 8% (added on air-dry basis) rapeseed oil (RSO). The POFA group produced milk with the highest fat content, and fat content was positively correlated with the mean size of milk fat globules. Goats in the RSO group had a higher content of long-chain and unsaturated fatty acids, whereas milk from goats in the POFA group had a higher content of palmitic and palmitoleic acids (C16:0 and C16:1 cis). The control group produced milk with a higher content of short-, medium-, odd-, and branched-chain fatty acids compared with the 2 other groups. The content of FFA in milk was low in early and late lactation and peaked in mid lactation (90 DIM). A high content of FFA was correlated with poor sensory properties (tart/rancid flavor). The RSO group produced milk with lower content of FFA and off-flavors in mid lactation and a higher proportion of unsaturated fatty acids. Therefore, replacement of palm oil with rapeseed oil as a lipid source in dairy goat feed would be favorable.

Dietary L-arginine supplementation differentially regulates expression of lipid-metabolic genes in porcine adipose tissue and skeletal muscle.

Obesity is a major health crisis worldwide and new treatments are needed to fight this epidemic. Using the swine model, we recently reported that dietary L-arginine (Arg) supplementation promotes muscle gain and reduces body-fat accretion. The present study tested the hypothesis that Arg regulates expression of key genes involved in lipid metabolism in skeletal muscle and white adipose tissue. Sixteen 110-day-old barrows were fed for 60 days a corn- and soybean-meal-based diet supplemented with 1.0% Arg or 2.05% L-alanine (isonitrogenous control). Blood samples, longissimus dorsi muscle and overlying subcutaneous adipose tissue were obtained from 170-day-old pigs for biochemical studies. Serum concentrations of leptin, alanine and glutamine were lower, but those for Arg and proline were higher in Arg-supplemented pigs than in control pigs. The percentage of oleic acid was higher but that of stearic acid and linoleic acid was lower in muscle of Arg-supplemented pigs, compared with control pigs. Dietary Arg supplementation increased mRNA levels for fatty acid synthase in muscle, while decreasing those for lipoprotein lipase, glucose transporter-4, and acetyl-coenzyme A carboxylase-α in adipose tissue. Additionally, mRNA levels for hormone sensitive lipase were higher in adipose tissue of Arg-supplemented pigs compared with control pigs. These results indicate that Arg differentially regulates expression of fat-metabolic genes in skeletal muscle and white adipose tissue, therefore favoring lipogenesis in muscle but lipolysis in adipose tissue. Our novel findings provide a biochemical basis for explaining the beneficial effect of Arg in improving the metabolic profile in mammals (including obese humans).

Effect of conjugated linoleic acid supplementation on lipoprotein lipase activity in 3T3-L1 adipocyte culture

Supplementation with conjugated linoleic acid may reduce fat body mass and increase lean body mass in various species. Some studies have demonstrated that conjugated linoleic acid reduces body fat, in part, by inhibiting the activity of lipoprotein lipase in adipocytes. The objective of this work was to study the effect of conjugated linoleic acid supplementation on lipoprotein lipase activity in 3T3-L1 adipocyte culture. 3T3-L1 adipocytes received linoleic acid (group C) or conjugated linoleic acid (group AE, supplemented with AdvantEdge® CLA, and group CO, supplemented with CLA One®) in concentrations of 1 mmol/L. Heparin-releasable lipoprotein lipase activity was analyzed by means of a 3T3-L1 adipocyte culture. After 7 days, heparin-releasable lipoprotein lipase activity was lower in the groups AE and CO supplemented with conjugated linoleic acid. These results suggest that one of the mechanisms by which CLA is capable of reducing body fat is by reducing lipoprotein lipase activity.

A suplementação com ácido linoléico conjugado pode reduzir a gordura corporal e aumentar a massa magra em diferentes espécies. Alguns estudos têm demonstrado que o ácido linoléico conjugado reduz a gordura corporal, por meio da inibição da atividade de lípase lipoprotéica em adipócitos. O objetivo deste estudo foi avaliar o efeito da suplementação com uma mistura de isômeros do ácido linoléico conjugado sobre a atividade da lípase lipoprotéica em cultura de adipócitos 3T3-L1. Os adipócitos 3T3-L1 receberam ácido linoléico (grupo controle) ou ácido linoléico conjugado (grupo AE, suplementado com AdvantEdge® CLA, e grupo CO, suplementado com CLA One®) na concentração de 1 mmol/L. A atividade de lípase lipoprotéica livre de heparina foi analisada pela média da cultura de adipócitos. Após 7 dias, a atividade da lípase lipoprotéica livre de heparina mostrou menores valores nos grupos AE e CO, suplementados com ácido linoléico conjugado. Estes resultados sugerem que um dos mecanismos pelo qual o ácido linoléico conjugado seja capaz de reduzir a gordura corporal é a partir da redução da atividade da lípase lipoprotéica.

Beneficiary effect of Tinospora cordifolia against high-fructose diet induced abnormalities in carbohydrate and lipid metabolism in Wistar rats.

High intake of dietary fructose has been shown to exert a number of adverse metabolic eff ects in humans and experimental animals. The present study was designed to investigate the eff ect of the aqueous extract of Tinospora cordifolia stem (TCAE) on the adverse eff ects of fructose loading toward carbohydrate and lipid metabolism in rats. Adult male Wistar rats of body weight around 200 g were divided into four groups, two of which were fed with starch diet and the other two with high fructose (66 %) diet. Plant extract of TC (400 mg/kg/day) was administered orally to each group of the starch fed rats and the highfructose fed rats. At the end of 60 days of experimental period, biochemical parameters related to carbohydrate and lipid metabolism were assayed. Hyperglycemia, hyperinsulinemia, hypertriglyceridemia, insulin resistance, and elevated levels of hepatic total lipids, cholesterol, triglycerides, and free fatty acids (p < 0.05) observed in fructose-fed rats were completely prevented with TCAE treatment. Alterations in the activities of enzymes of glucose metabolism (hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, and glucose-6-phosphate dehydrogenase) and lipid metabolism (fatty acid synthetase, lipoprotein lipase, and malic enzyme) as observed in the high fructose-fed rats were prevented with TCAE administration. In conclusion, our fi ndings indicate improvement of glucose and lipid metabolism in high-fructose fed rats by treatment with Tinospora cordifolia, and suggest that the plant can be used as an adjuvant for the prevention and/or management of insulin resistance and disorders related to it.

trans-10,cis-12 Conjugated linoleic acid inhibits lipoprotein lipase but increases the activity of lipogenic enzymes in adipose tissue from hamsters fed an atherogenic diet.

The aim of the present work was to investigate the effects of trans-10,cis-12 conjugated linoleic acid (CLA) on the activity and expression of lipogenic enzymes and lipoprotein lipase (LPL), as well as on the expression of transcriptional factors controlling these enzymes, in adipose tissue from hamsters, and to evaluate the involvement of these changes in the body fat-reducing effect of this CLA isomer. Thirty male hamsters were divided into three groups and fed atherogenic diets supplemented with 0 (linoleic group), 5 or 10 g trans-10,cis-12 CLA/kg diet, for 6 weeks. Body and adipose tissue weights, food intake and serum insulin were measured. Total and heparin-releasable LPL and lipogenic enzyme activities (acetyl-CoA carboxylase (ACC); fatty acid synthase (FAS); glucose-6-phosphate dehydrogenase (G6PDH); and malic enzyme (ME)) were assessed. ACC, FAS, LPL, sterol regulatory element-binding proteins (SREBP-1a), SREBP-1c and PPARgamma mRNA levels were also determined by real-time PCR. CLA did not modify food intake, body weight and serum insulin level. CLA feeding reduced adipose tissue weight, LPL activity and expression, and increased lipogenic enzyme activities, despite a significant reduction in ACC and FAS mRNA levels. The expression of the three transcriptional factors analysed (SREBP-1a, SREBP-1c and PPARgamma) was also reduced. These results appear to provide a framework for partially understanding the reduction in body fat induced by CLA. Inhibition of LPL activity seems to be an important mechanism underlying body fat reduction in hamsters. Further research is needed to better characterize the effects of CLA on lipogenesis and the role of these effects in CLA action.

Genetic factors associated with response of LDL subfractions to change in the nature of dietary fat.

A preponderance of dense low density lipoprotein (LDL) particles is associated with an increased risk of coronary heart disease. It has been shown that dense LDL levels can be modified by diet. We investigated the contribution of polymorphisms in the genes for apolipoprotein (apo) B, apo AIV, lipoprotein lipase (LPL) and cholesterol ester transfer protein (CETP) to variation in the changes in plasma concentrations of dense LDL between a high saturated and a high polyunsaturated fatty acid diet. A total of 46 freeliving individuals (19 men and 27 women) completed a crossover trial with two dietary interventions of 4 weeks each, a high saturated fat diet (providing 21% energy from saturated fat and 3% energy from polyunsaturated fat) and a high polyunsaturated fat diet (providing 11% energy as saturated fat and 10% energy as polyunsaturated fat). Overall, the change in dense LDL between the saturated and polyunsaturated fat period was 0.17+/-0.33 mmol/L and this change was similar in men and women. Of the polymorphisms studied only variation in the apo AIV gene causing the substitution of histidine for glutamine at position 360 (Q360H) was associated with significant differences in the change in dense LDL concentration. Apo AIV Q/H individuals (n=6) showed a three-fold greater change in dense LDL cholesterol unadjusted for Lp(a) levels than Q/Q individuals (0.46+/-0.27 versus 0.12+/-0.31 mmol/L, p=0.02). The greater decrease in dense LDL cholesterol with an increase in polyunsaturated fat seen in those with the apo AIV H360 variant, who represent roughly 10% of the general population, suggests that they may benefit most from a PUFA rich lipid lowering diet.

Alterations in cholesteryl ester transfer, lipoprotein lipase, and lipoprotein composition after combined pancreas-kidney transplantation.

IDDM patients treated with conventional subcutaneous insulin have an abnormal increase in cholesteryl ester transfer (CET), the proatherogenic step in reverse-cholesterol transport that results in the enrichment of the apolipoprotein (apo) B-containing lipoproteins (VLDL, LDL) with cholesteryl ester (CE). This disturbance is closely linked to iatrogenic hyperinsulinemia and the nonphysiologic stimulation of lipoprotein lipase (LpL), a physiologic activator of CET, because lowering systemic insulin levels by administering insulin through the intraperitoneal insulin route normalizes LpL and CET. Hyperinsulinemia persists in IDDM patients who undergo successful pancreas-kidney transplantation (PKT) when their allografts are placed in the pelvis and drain into the iliac vein. Therefore, to determine whether hyperinsulinemia promotes CET in this setting, we studied CET, LpL, and insulin levels in 14 euglycemic normolipidemic IDDM PKT patients with near-normal kidney function (creatinine 1.5 +/- 0.4 mg/dl). Consistent with our prediction, the net mass of CE transferred from HDL to VLDL + LDL was significantly increased in the PKT group (P < 0.001) compared with nondiabetic renal transplant patients receiving the same immunosuppressive drugs and healthy control subjects. Both basal and arginine-stimulated insulin levels were increased above the kidney transplant group's levels and correlated with the mass of CE transferred at 2 h (r = 0.71, P < 0.05; r = 0.66, P < 0.05, respectively). Total basal LpL activities, LpL and hepatic triacylglycerol lipase activities, and LpL mass all tended to be higher than levels in healthy control subjects. Consistent with these changes in lipase activity, VLDL particle size was significantly reduced (P < 0.025) compared with that of control subjects. These findings indicate that PKT patients with systemically draining allografts have a persisting profile of potentially atherogenic disturbances in insulin levels, LpL, and CET that resemble IDDM patients treated with conventional subcutaneous insulin injections.

Temporal-regulation of serum lipids and stearoyl CoA desaturase and lipoprotein lipase mRNA in BALB/cHnn mice.

Databases for genes expressed in humans or cell cultures are being developed as a part of the Human Genome Project. Because genomes respond to nutritional and other environmental variables, quantitative analyses of mRNA abundance under defined nutritional and physiological states are required to understand normal metabolism and to clarify differences between normal and disease phenotypes. Reported here are comparisons of food intake, growthp5erum lipids and expression of mRNA for hepatic stearoyl CoA desaturase (Scd1) and heart lipoprotein lipase (Lpl) in female BALB/cHnn mice following food deprivation and refeeding at the end of 2 wk of feeding semipurified diets with 3, 10 or 20% corn oils. Body weights and utilization of dietary energy were similar for mice fed all three diets. There were no differences in serum lipid concentrations associated with the level of dietary fat during subsequent food deprivation and refeeding, but significant differences in serum triglycerides and total serum cholesterol were observed between food-deprived and fed mice. Heart lipoprotein lipase and hepatic Scd1 mRNA expression levels were affected significantly by concentration of corn oil and by time after eating. These and other studies examining gene regulation by dietary variables and nutrient availability are discussed in relation to development of diet-regulated gene databases for laboratory animals fed semipurified diets.

Serum triiodothyronine concentration and Na+,K(+)-ATPase activity in liver and skeletal muscle are influenced by dietary fat type in rats.

It has been shown previously that dietary fat type influences body fat accumulation in rats. The effects of dietary fat type on serum thyroid hormone, activity of Na+,K(+)-ATPase and lipoprotein lipase were studied. Rats were fed an experimental diet containing lard, high oleic safflower oil, safflower oil or linseed oil for 12 wk. Carcass fat content was significantly higher in rats fed the lard diet than in those fed the other diets. However, intra-abdominal adipose tissue weights were not affected by type of dietary fat. The serum triiodothyronine concentration and the activity of Na+,K(+)-ATPase in the liver and skeletal muscle were significantly lower in the lard diet group than in the other diet groups. The lipoprotein lipase activity of abdominal subcutaneous fat was significantly higher in rats fed the lard diet than in rats fed the other diets, but the activity of lipoprotein lipase in intra-abdominal fat was not significantly different. These results suggest that the intake of lard, compared with the intake of the vegetable oils, may decrease Na+,K(+)-ATPase activity in the liver and skeletal muscle by lowering serum triiodothyronine concentration, resulting in the promotion of body fat accumulation.

Hypoalphalipoproteinemia: postprandial response of subjects with preprandial normotriglyceridemia and hypertriglyceridemia to various diets.

The effect of a single oral fat meal (60 g fat/m2 body surface area) enriched in either saturated (SFA) or polyunsaturated ([PUFA] omega-6 or omega-3) fatty acids on postprandial lipoprotein levels was studied in four men with primary hypoalphalipoproteinemia (HP) and in four age- and sex-matched controls. Vitamin A was included in the meal to label intestinally derived triglyceride-rich particles (TRP) with retinyl palmitate (RP). The HP subjects were either mildly hypertriglyceridemic (group A) or normotriglyceridemic (group B) and were phenotyped for post-heparin lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) activities and apolipoprotein (apo) E isoforms. Postprandial total plasma triglyceride (TG), high-density lipoprotein (HDL) cholesterol, and RP and TG concentrations in the chylomicron (Sf > 1,000) and nonchylomicron (Sf > 1,000) fractions were evaluated for 24 hours after the meal. At each time point, HDL composition and size and apolipoprotein distributions were also measured. Following the SFA meal, HP subjects had maximal plasma TG levels at 8 hours (4 hours in controls) with a slow return to baseline levels at 12 to 24 hours (8 to 12 hours for controls). In contrast, after the omega-6 meal plasma TG levels decreased in group A subjects, while group B subjects and controls showed only a small increase. The results after the omega-6 meal were intermediate between the SFA and the omega-3 meal. When compared with group B, subjects in group A showed higher levels of RP-associated TRP, slower clearance rates, 30% to 50% lower fasting LPL activity, and 1.5-fold to twofold higher fasting plasma apo C-III levels. The major preprandial HDL subclass in HP subjects was HDL3, which showed a relative decrease in cholesterol esters (CE) and an increase in TG levels following the SFA meal. After the omega-3 meal, HDL of group A subjects showed a decrease in TG, a reciprocal increase in CE, and either no changes or minor changes in phospholipid (PL) and free-cholesterol (FC) levels. The results show that HP subjects with mild preprandial hypertriglyceridemia respond to a single fat meal differently than subjects with normotriglyceridemia, and that this difference is the result of HP in addition to other factors such as low LPL and HTGL activities, high plasma apo C-III levels, and apo E2 phenotype.

Dependence on Ca2+ of lipoprotein lipase stability.

Macrophages continuously secrete lipoprotein lipase (LPL) into the culture medium. When LPL was collected from thioglycollate-elicited peritoneal macrophages (Tg-Mø) or J774.1 cells over a 4 h period in Ca2+ and Mg2(+)-free Dulbecco's modified Eagle's medium (d-DMEM) the activity in the collection medium was reduced by 40-62% and 23%, respectively, as compared to that expressed in full medium (DMEM). Ca2+ supplementation during the collection period in d-DMEM augmented LPL activity in the medium; about 1 mM Ca2+ was required for attainment of activity comparable to that expressed in DMEM. Addition of Ca2+ during the assay did not enhance LPL activity collected into d-DMEM. Addition of EGTA to the assay mixture reduced LPL activity by 34-60% and when present in the collection medium, EGTA led to a reduction in enzyme activity greater than 90%. A 4 h incubation of Tg-Mø in 3 mM EGTA led to an almost complete loss of intracellular Ca2+ (measured by efflux of 45Ca2+ from preloaded cells), yet there was no change in the overall synthesis and secretion of proteins and in the phagocytic capability of the cells. LPL activity in the enzyme collection medium after its removal from cell monolayers was stable at least up to 4 h at 0 degrees C and at 23 degrees C. Activity was progressively lost with increased temperatures: up to 40% loss at 37 degrees C in 4 h. Addition of EGTA to the above medium led to an enhanced rate of irreversible enzyme inactivation: 76-86% loss of activity in 4 h at 37 degrees C. No inactivation was observed at 0 degrees C and at 23 degrees C in the presence of EGTA. The results indicate a critical role for Ca2+ in enzyme stabilization.

Paracellular leakage of lipoprotein lipase across the mammary epithelium of the goat.

The ability of lipoprotein lipase to move across the mammary epithelium by a paracellular route was investigated. Five goats were milked hourly to activate the paracellular pathway. Three goats responded to hourly milking with a fivefold increase in milk lipoprotein lipase activity as compared with nonresponding goats. Massage of the mammary gland was necessary in the two nonresponding goats too cause increased lipoprotein lipase activity in milk. Oxytocin treatment during hourly milking also increased enzyme activity in milk from a nonresponding goat. Activation of the paracellular pathway by hourly milking increased milk sodium and protein and decreased potassium and lactose concentrations. After a 12-h milking interval, lipoprotein lipase activity was distributed primarily in the serum (48%) and cream (40%) fractions and, to a lesser extent, in the casein (12%) fraction. Hourly milking increased enzyme activity distributed in the serum fraction (62%), whereas enzyme activity associated with the cream (32%) and casein (6%) fractions decreased. Possible mechanisms for the origin of lipoprotein lipase in milk are discussed.

Human milk lipases. I. Serum-stimulated lipase.

Lipase activity has previously been demonstrated in human milk. This study shows that there are two separate triglyceride lipases in human milk. One is mainly in the skim milk and is stimulated by bile salts; the other is mainly in the cream and is inhibited by bile salts but stimulated by serum. The serum-stimulated lipase was purified by affinity chromatography on heparin-substituted Sepharose 4B. This gave a 9500-fold purification over whole milk. Although polyacrylamide gel electrophoresis showed that the enzyme was not purified to homogeneity, it had the highest specific activity so far reported for a human serum-stimulated lipase. The purified enzyme was free from bile salt-stimulated lipase activity and had the characteristics of other serum-stimulated or so-called lipoprotein lipases. Thus, it was almost completely inhibited by 1 M NaCl. The purified enzyme was active against tributyrylglycerol also in the absence of exogenous serum factors.