Evaluation of the physicochemical and biological stability of reconstituted and diluted SB2 (infliximab).

OBJECTIVES: To evaluate the critical quality attributes that might affect the stability of an infliximab biosimilar (SB2, Flixabi) when reconstituted or diluted and stored under refrigeration and at room temperature. METHODS: We largely adhered to the UK's National Health Service guidance requirements for the design of a robust stability study and for robust testing methods. Protocol components included evaluation of visual appearance, chemical stability, physical stability, pH, particle sizes and biological activity. The stability of reconstituted SB2 was assessed for 60 days at 5°C and for 7 days at 25°C. Stability of diluted SB2 at concentrations that ranged from 240 mg/250 mL (3 mg/kg; 80 kg patient) to 400 mg/250 mL (5 mg/kg; 80 kg patient) was assessed for 7 days at both temperatures. Dilutions were made in polyethylene bags containing 0.9% NaCl. Forced degradation studies were conducted with SB2 and its reference product (USA-sourced and European Union-sourced Remicade). Stress conditions of heat or light occurred before product reconstitution. RESULTS: In a laboratory environment under aseptic conditions, stability acceptance criteria with regard to physicochemical and biological properties were met for all reconstituted and diluted SB2 samples for all time periods and temperatures assessed. After either heat or light stress, similar stability and biological activity were noted for SB2 and both reference products. CONCLUSIONS: When prepared under aseptic conditions in accordance with the product's Summary of Product Characteristics, exposed for prolonged periods at 5°C and 25°C and assessed with the described methods, SB2 appears to remain a stable monoclonal antibody maintaining its expected biological function.

Comprehensive Physicochemical and Biological Characterization of the Proposed Biosimilar Darbepoetin Alfa, LBDE, and Its Originator Darbepoetin Alfa, NESP®.

BACKGROUND: For regulatory approval, the comparability of a biosimilar product to an originator product should be ensured through thorough physicochemical and biological characterization. OBJECTIVE: To evaluate the biosimilarity between LBDE, the proposed biosimilar darbepoetin alfa, and NESP®, its originator, we performed a comprehensive physicochemical and biological characterization study. METHODS: Primary and higher-order protein structures were analyzed using Lys-C peptide mapping with liquid chromatography-mass spectrometry (LC-MS), disulfide bond identification, circular dichroism, and fluorescence spectroscopy. Glycosylation and isoform distribution were analyzed using MS, LC, and capillary zone electrophoresis. Size variants were evaluated with size-exclusion chromatography-high-performance liquid chromatography (SEC-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Biological characterization included binding affinity for human erythropoietin receptor, in vitro cell proliferation, and in vivo potency. Pharmacokinetics (PK) were evaluated using rats through two injection routes. RESULTS: Non-reducing and reducing Lys-C peptide mapping showed a highly similar peak profile, confirming that LBDE and NESP® have the same primary structure and disulfide bonds. Glycosylation and isoform analyses showed that the attached N-glycan and O-glycan structures were the same and their relative contents were similar. Spectroscopic analysis of LBDE showed indistinguishable spectra with NESP®. For both LBDE and NESP®, a very small amount of size variants was found in SEC-HPLC, and no minor bands were detected in SDS-PAGE. Furthermore, LBDE did not show any difference with NESP® in the in vitro and in vivo functional analyses. PK parameters of LBDE were in good agreement with those of NESP®. CONCLUSION: LBDE shows high similarity to NESP® with regard to structure and function.

Associations between metabolomic-identified changes of biomarkers and arterial stiffness in subjects progressing to impaired fasting glucose.

OBJECTIVE: We investigated correlations between age-related changes in circulating metabolites and arterial stiffness in impaired fasting glucose (IFG). DESIGN, SUBJECTS, MEASUREMENT: This prospective cohort study included 602 healthy, normal fasting glucose (NFG) subjects (30-65 years old) who underwent triennial medical evaluation. After 3 years, 9·3% of subjects developed IFG (n = 56). Age, gender, BMI and fasting glucose were used to match the remaining NFG subjects (n = 546) that were included for the control group (NFG group, n = 80). RESULTS: After 3 years, levels of fasting glucose, insulin and malondialdehyde, and brachial-ankle pulse wave velocity (baPWV) were significantly greater in the IFG group than in the NFG group after adjusting for baseline values. The IFG group had a greater increase in lactosylceramide (P = 0·001, q < 0·05) and a greater reduction in phosphatidylcholine (PC) (18:0/20:4) than the NFG group. Multiple linear regression analysis showed that the change in baPWV was independently and positively associated with changes in fasting glucose and lactosylceramide. In all subjects, lactosylceramide levels positively correlated with changes in baPWV and fasting glucose, while premenopausal women were not shown, and negatively correlated with changes in PC and LDL particle size. CONCLUSIONS: This study indicates that age-related increase in circulating lactosylceramide is an independent predictor of increased arterial stiffness in subjects with impaired fasting glucose.

Altered plasma lysophosphatidylcholines and amides in non-obese and non-diabetic subjects with borderline-to-moderate hypertriglyceridemia: a case-control study.

Hypertriglyceridemia (HTG) is a risk factor for atherosclerotic cardiovascular disease (CVD). We investigated alterations in plasma metabolites associated with borderline-to-moderate HTG (triglycerides (TG) 150-500 mg/dL). Using UPLC-LTQ-Orbitrap mass spectrometry analysis, the metabolomics profiles of 111 non-diabetic and non-obese individuals with borderline-to-moderate HTG were compared with those of 111 age- and sex-matched controls with normotriglyceridemia (NTG, TG <150 mg/dL). When compared to the NTG control group, the HTG group exhibited higher plasma levels of lysophosphatidylcholines (lysoPCs), including C14:0 (q = 0.001) and C16:0 (q = 1.8E-05), and several amides, including N-ethyldodecanamide (q = 2.9E-05), N-propyldodecanamide (q = 3.5E-05), palmitoleamide (q = 2.9E-06), and palmitic amide (q = 0.019). The metabolomic profiles of the HTG group also exhibited lower plasma levels of cis-4-octenedioic acid (q<1.0E-9) and docosanamide (q = 0.002) compared with those of the NTG controls. LysoPC 16:0 and palmitoleamide emerged as the primary metabolites able to discriminate the HTG group from the NTG group in a partial least-squares discriminant analysis and were positively associated with the fasting triglyceride levels. We identified alterations in lysoPCs, amides, and cis-4-octenedioic acid among non-diabetic and non-obese individuals with borderline-to-moderate HTG. These results provide novel insights into the metabolic alterations that occur in the early metabolic stages of HTG. This information may facilitate the design of early interventions to prevent disease progression.

Association between arterial stiffness and serum L-octanoylcarnitine and lactosylceramide in overweight middle-aged subjects: 3-year follow-up study.

Existing data on the association between being overweight and cardiovascular morbidity and mortality risk in adults are inconsistent. We prospectively and longitudinally investigated the effects of weight on arterial stiffness and plasma metabolites in middle-aged subjects (aged 40-55 years). A group of 59 individuals who remained within the range of overweight during repeated measurements over a 3-year period was compared with a control group of 59 normal weight subjects who were matched for age and gender. Changes in metabolites by UPLC-LTQ-Orbitrap mass spectrometry and changes in brachial-ankle pulse wave velocity (ba-PWV) were examined. At baseline, the overweight group showed higher BMI, waist circumference, triglyceride, free fatty acid (FFA), glucose, insulin, and hs-CRP, and lower HDL-cholesterol than controls. After 3 years, the changes in waist circumference, diastolic and systolic blood pressure (DBP and SBP), triglyceride, FFA, glucose, insulin, hs-CRP, and ba-PWV observed in the overweight group were significantly different from those in the control group after adjusting for baseline levels. Furthermore, the overweight group showed greater increases in L-octanoylcarnitine (q=0.006) and decanoylcarnitine (q=0.007), and higher peak intensities of L-leucine, L-octanoylcarnitine, and decanoylcarnitine. Multiple linear regression analysis showed that the change in ba-PWV was independently and positively associated with changes in L-octanoylcarnitine, lactosylceramide, and SBP, and with baseline BMI. Our results indicate that the duration of overweight is an important aggravating factor for arterial stiffness, especially during middle age. Additionally, an age-related increase in plasma L-octanoylcarnitine, lactosylceramide, SBP, and baseline BMI are independent predictors of increased arterial stiffness in middle-aged individuals.

Carriage of the V279F homozygous genotype, a rare allele, within the gene encoding Lp-PLA2 leads to changes in circulating intermediate metabolites in individuals without metabolic syndrome.

AIM: Identifying differences in plasma metabolic profiling between Lp-PLA2 279VV and 279FF in individuals without metabolic syndrome (MetS) can be used to elucidate the roles of novel Lp-PLA2 activities in normal physiological processes. METHODS: Non-MetS individuals with 279FF (n=36) and age-, sex- and BMI-matched VV subjects (n=36) were included in this analysis. RESULTS: The FF subjects exhibited no appreciable enzyme activity. No significant differences were observed between the VV and FF subjects in the serum lipid profiles or hs-CRP, plasma ox-LDL, MDA or urinary 8-epi-PGF2α levels. The FF subjects also showed lower activities of lyso-phosphatidylcholine (lysoPC) (16:0) (p=0.003) and oleamide (p＜0.001) and a higher activity of L-tryptophan (p=0.016) than the VV subjects. In addition, the Lp-PLA2 activity positively correlated with the lysoPC (16:0) and lysoPC (18:0) activities and negatively correlated with the PC (16:0/22:6) and L-tryptophan activities in the VV subjects. Furthermore, in the VV subjects, the lysoPC (16:0) and lysoPC (18:0) activities negatively correlated with the presence of PCs containing 14:0/20:2, 14:0/22:4 and 16:0/22:6, while the oleamide activity exhibited a strong positive correlation with lysoPCs and a negative correlation with PCs, whereas the relationship between oleamide and lysoPCs and PCs was weaker in the FF subjects. CONCLUSIONS: The present results indicate that the natural absence of the plasma Lp-PLA2 activity due to carriage of the Lp-PLA2 279FF genotype may reduce the generation of lysoPC (16:0) and oleamide and thereby enhance the activity of plasma tryptophan in normal physiological processes.

Age-related increase in alanine aminotransferase correlates with elevated levels of plasma amino acids, decanoylcarnitine, Lp-PLA2 Activity, oxidative stress, and arterial stiffness.

We investigated plasma metabolite profiles that correlated with age-related serum alanine aminotransferase (ALT) levels. The study included 602 healthy, nondiabetic subjects (aged 30-65 years); 393 individuals had normal ALT levels at baseline. Fifty-three (13.5%) individuals developed elevated ALT levels after 3 years. The remaining 340 subjects with normal ALT were matched to the elevated-ALT group (n = 53) for age, gender, BMI, fasting glucose, and ALT to form the control group (n = 53). At the 3-year follow-up, the elevated-ALT group exhibited greater increases in waist circumference, serum free fatty acid, ALT, aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), bilirubin, plasma oxidized LDL, Lp-PLA2 activity, urinary 8-epi-prostaglandin F2α (8-epi-PGF2α), and brachial-ankle pulse-wave velocity (ba-PWV) compared to the control group after baseline adjustment. The elevated-ALT group exhibited greater increases in plasma l-valine (q = 0.036), l-leucine (q = 0.012), l-phenylalanine (q = 0.012), and decanoylcarnitine (q = 0.002). Mean ALT levels positively correlated with changes in these four metabolites, which correlated with changes in AST, GGT, Lp-PLA2 activity, urinary 8-epi-PGF2α, and ba-PWV. Mean ALT changes did not significantly correlate with HOMA-insulin resistance. These results suggest that increased plasma levels of l-valine, l-leucine, l-phenylalanine, and decanoylcarnitine precede insulin resistance during periods of elevated ALT. This metabolic disturbance coincides with enhanced risk factors for cardiovascular disease.

Prehypertension-associated elevation in circulating lysophosphatidlycholines, Lp-PLA2 activity, and oxidative stress.

Prehypertension is a risk factor for atherosclerosis. We investigated alterations in plasma metabolites that are associated with prehypertension. A group of 53 individuals was identified who remained within the range of prehypertension during repeated measurements in a 3-year period. This group was compared with the control group of 53 normotensive subjects who were matched for age and gender. Metabolomic profiles were analyzed with UPLC-LTQ-Orbitrap mass spectrometry. The prehypertensive group showed higher levels of lysophosphatidylcholines (lysoPCs) containing C14:0, C16:1, C16:0, C18:2, C18:1, C18:0, C20:5, C20:4, C20:3, and C22:6, higher circulating Lp-PLA2 activity, oxidized LDL (ox-LDL), interleukin 6 (IL-6), urinary 8-epi-PGF2α, and higher brachial-ankle pulse wave velocity (ba-PWV), before and after adjusting for BMI, WHR, smoking, alcohol consumption, serum lipid profiles, glucose, and insulin. LysoPC (16:0) was the most important plasma metabolite for evaluating the difference between control and prehypertensive groups, with a variable important in the projection (VIP) value of 17.173, and it showed a positive and independent association with DBP and SBP. In the prehypertensive group, the levels of lysoPC (16:0) positively and significantly correlated with ox-LDL, Lp-PLA2 activity, 8-epi-PGF2α, ba-PWV, and IL-6 before and after adjusting for confounding variables. Prehypertension-associated elevations in lysoPCs, Lp-PLA2 activity, ox-LDL, urinary 8-epi-PGF2α, IL-6, and ba-PWV could indicate increased oxidative stress from Lp-PLA2-catalyzed PC hydrolysis during increased LDL oxidation, thereby enhancing proinflammation and arterial stiffness.

Characterization of thiol-specific antioxidant 1 (TSA1) of Candida albicans.

We previously identified several proteins that are differentially expressed in pathogenic hyphae by comparing protein profiles of yeast and hyphae of Candida albicans. One of these, thiol-specific antioxidant 1 (TSA1), attracted our attention because it may play some roles in surviving an unfavourable oxidative environment created by host cells. Two alleles of the C. albicans TSA1 (CaTSA1) gene are located in opposite orientation on the same chromosome. Using PCR-directed disruption cassettes and URA-Blaster, a series of deletion mutants that lack one to four copies were constructed to examine the functions of CaTSA1. Northern and Western analyses showed that both the transcript and protein products of CaTSA1 decreased proportionally to the disrupted copy number and were completely absent in the null mutant, indicating that all four TSA1 copies are equally functional at the transcriptional level. Intracellular H2O2 increased by an order of magnitude in deletion mutants lacking three to four copies, suggesting that CaTsa1p is not a redundant H2O2 scavenger. CaTsa1p was indispensable for yeast-to-hyphal transition when C. albicans was cultured under oxidative stress. The level of its oxidized form increased approximately five-fold in hyphal cells, whereas that of the reduced form increased two-fold compared to yeast cells. The ratio of oxidized to reduced form was increased three-fold in hyphal cells. This overall increase was found to be controlled at the post-transcriptional level. Interestingly, CaTsa1p is translocated to the nucleus of hyphal cells. These findings may be of biological significance for differentiation and pathogenicity.