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In the first part of this publication, the results from an international study evaluating the precision (i.e., repeatability and reproducibility) of N-glycosylation analysis using capillary electrophoresis of APTS-labeled N-glycans were presented. The corresponding results from ultra-high performance liquid chromatography (UHPLC) with fluorescence detection are presented here from 12 participating sites. All participants used the same lot of samples, reagents, and columns to perform the assays. Elution time, peak area and peak area percent values were determined for all peaks ≥0.1% peak area, and statistical analysis was performed following ISO 5725-2 guideline principles. The results demonstrated adequate reproducibility, within any given site as well across all sites, indicating that standard UHPLC-based N-glycan analysis platforms are appropriate for general use., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established.

The term "biosimilar" is used to designate a follow-on biologic that meets extremely high standards for comparability or similarity to the originator biologic drug that is approved for use in the same indications. Use of biosimilar products has already decreased the cost of treatment in many regions of the world and now a regulatory pathway for approval of these products has been established both in US and in the EU. The analytical tests to demonstrate comparability and similarity of a biosimilar product to a reference drug with respect to protein content, activity, physiochemical integrity, stability, impurities and additives, as well as immunogenicity are discussed., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In this paper, the effect of temperature is investigated on the performance of glycoprotein enrichment by boronic acid lectin affinity chromatography (BLAC). Wheat germ agglutinin and m-aminophenyl boronic acid containing stationary phases were evaluated individually and in a mixed mode using an automated liquid handling robot with an integrated 96-well plate temperature controller. Glycoaffinity enrichment of the model proteins of ribonuclease B and trypsin inhibitor was investigated in the presence of the non-glycosylated proteins of myoglobin (neutral) and lysozyme (basic) at a wide temperature range of 5-65 degrees C. Our results revealed that glycoaffinity micropartitioning at the temperature of 25 degrees C provided the highest recovery rate for glycoprotein enrichment. We have also found that a large amount of lysozyme was present in the elution fractions of the m-aminophenyl boronic acid containing micropartitioning columns due to ion-exchange mechanism occurring between the positively charged protein and the negatively charged stationary phase at the operation pH. On the other hand, at high temperature (65 degrees C), non-specific interactions with the agarose carrier prevailed, evidenced by the presence of myoglobin in the eluate.

As a continuation of our work on boronic acid lectin affinity chromatography (BLAC), in this paper we introduce an automated affinity micropartitioning approach using combined boronic acid and concanavalin A (BLAC/Con A) resin-filled micropipette tips to isolate and enrich human serum glycoproteins. The N-linked oligosaccharides of the partitioned glycoproteins were removed by PNGase F enzyme digestion, followed by 8-aminopyrene-1,3,6-trisulfonic acid labeling. Capillary gel electrophoresis with blue LED-induced fluorescence detection was applied in a multiplexed format for comparative glycan profiling. The efficiency of BLAC affinity micropartitioning was compared with that of the individual lectin and pseudolectin affinity enrichment. Finally, we report on our findings in glycosylation differences in human serum samples from healthy and prostate cancer patients by applying BLAC/Con A micropipette tip-based enrichment and comparative multicapillary gel electrophoresis analysis of the released and labeled glycans.

This paper evaluates various sample preparation methods for multicapillary gel electrophoresis based glycan analysis to support electrokinetic injection. First the removal of excess derivatization reagent is discussed. Although the Sephadex G10 filled multiscreen 96-well filter plate and Sephadex G10 filled pipet tips enabled increased analysis sensitivity, polyamide DPA-6S pipet tips worked particularly well. In this latter case an automated liquid handling system was used to increase purification throughput, necessary to feed the multicapillary electrophoresis unit. Problems associated with the high glucose content of such biological samples as normal human plasma were solved by applying ultrafiltration. Finally, a volatile buffer system was developed for exoglycosidase-based carbohydrate analysis.

The stochastic theory of chromatography and an equilibrium based approach were used for the prediction of peak shape and retention data of anions. This attempt incorporating the potential advantages of two different chromatographic phenomena for analytical purposes. It is an integrated method to estimate kinetic and thermodynamic properties for the same chromatographic run of ions. The stochastic parameters of eluted anions, such as the residence time of the molecule on the surface of the stationary phase, and the average number of adsorption steps were determined on the basis of a retention database of organic and inorganic anions (formate, chloride, bromide, nitrate, sulphate, oxalate, phosphate) obtained by using carbonate/bicarbonate eluent system at different pHs (9-11) and concentrations (7-13 mM). In the investigated IC system the residence times are much higher and the average number of sorption steps is somewhat smaller than in RP-HPLC. The simultaneous application of the stochastic and the multispecies eluent/analyte model was utilized to peak shape simulation and the retention controlling of various anions under elution conditions of practical importance. The similarities between the measured and the calculated chromatograms indicates the predictive and simulation power of the combined application of the stochastic theory and the multiple species eluent/analyte retention model.

To determine the extent of alpha 2-adrenoreceptor control of cardiovascular function, we studied the hemodynamic effects of the relatively selective alpha 2-adrenergic agonist UK 14,304-18 on the heart and peripheral circulation of intact dogs. Administration of increasing intravenous doses of UK 14,304-18 to conscious dogs given atropine to maintain heart rate (HR) resulted in a reproducible increase in mean aortic (AO) pressure (77.6 +/- 5.0 to 136.4 +/- 6.5 mm Hg, p less than 0.05) and reductions in stroke volume (31.7 +/- 2.9 to 17.9 +/- 1.9 ml/kg/min, p less than 0.05) and left ventricular (LV) dP/dt (2,120 +/- 280.0 to 1,463 +/- 196.1 mm Hg/s, p less than 0.05). In ganglion-blocked dogs UK 14,304-18 did not alter the slope of the LV end-systolic pressure-volume relationship when compared with angiotensin and nitroprusside (79.9 +/- 11.1 control vs. 73.3 +/- 8.7 mm Hg/ml/kg UK 14,304-18, p greater than 0.05), nor did it change the volume intercept (-0.46 +/- 0.12 control vs. -0.53 +/- 0.16 ml/kg UK 14, 304-18, p greater than 0.05) indicating no direct effect on LV contractile function. Changes in indices of diastolic function, including the time constant of isovolumic relaxation, time to peak filling, and chamber volume elasticity were similar to those of equipressor doses of angiotensin, indicating no direct effect on LV diastolic function. Effects on the peripheral circulation were studied in dogs undergoing transient acetylcholine-induced circulatory arrest. UK 14,304-18 increased mean circulatory filling pressure (7.9 +/- 0.3 to 10.3 +/- 0.2 mm Hg, p less than 0.05) and the pressure gradient for venous return (7.6 +/- 0.4 to 9.0 +/- 0.3 mm Hg, p less than 0.05). Central blood volume increased with UK 14,304-18 (15.6 +/- 1.1 to 18.7 +/- 1.5 ml/kg, p less than 0.05), but this increase was not sufficient to maintain cardiac output (CO) during the UK 14,304-18 infusion, which decreased from 157.4 +/- 11.1 to 131.5 +/- 8.9 ml/kg/min (p less than 0.01) in the presence of increased LV afterload. The time constant of relaxation of the arterial system increased and the arterial compliance decreased with increasing mean arterial pressure. Thus, this relatively selective alpha 2 agonist does not directly alter cardiac function but increased tone in arterial resistance vessels and in systemic veins. The fall in CO appears to be caused by a mismatch between preload and afterload, which is the net result of quantitatively different effects on systemic veins and arteries.

The purpose of this study was to define the effects of nitroglycerin on venous tone and to investigate the time course of nitroglycerin tolerance in the peripheral circulation. The changes in the arterial and venous circulation resulting from an intravenous infusion of nitroglycerin (5 micrograms/kg per min) after 5 minutes (acute infusion) were compared with those changes that occurred after 2 hours (chronic infusion) of the same infusion in six splenectomized, ganglion-blocked dogs. Hemodynamics, blood volume and venous and arterial compliance were measured during each infusion. Nitroglycerin initially decreased mean arterial pressure from 81.5 +/- 2.0 to 57.6 +/- 2.7 mm Hg (p less than 0.01). Central blood volume decreased from 21.1 +/- 1.4 to 15.9 +/- 1.1 ml/kg (p less than 0.01), while total blood volume and unstressed vascular volume did not change. In the acute study, nitroglycerin increased venous compliance 33% from 1.75 +/- 0.14 to 2.32 +/- 0.16 ml/mm Hg per kg (p less than 0.01) and arterial compliance 33% from 0.049 +/- 0.007 to 0.065 +/- 0.007 ml/mm Hg per kg (p less than 0.01). At the end of the 2 hour infusion, arterial pressure increased and was now unchanged from control. Central blood volume had returned to baseline, 17.8 +/- 0.9 ml/kg. Total blood volume and unstressed vascular volume remained unchanged. With the long-term infusion, both arterial and venous compliance decreased (p less than 0.02) to 0.050 +/- 0.006 and 1.50 +/- 0.06 ml/mm Hg per kg, respectively, such that neither value was different from control. Nitroglycerin levels remained constant throughout.(ABSTRACT TRUNCATED AT 250 WORDS)

The extent of cholinergic-sympathetic interaction in the heart was examined in the resting control state and after isoproterenol stimulation in eight conscious dogs. Sonomicrometer crystals and solid-state pressure transducers were implanted in the left atrium and the left ventricle to evaluate mechanical function while heart rate was held constant by atrial pacing. Edrophonium (0.5 mg/kg i.v.) was given as a single dose at rest and during increased sympathetic tone produced by continuous infusion of isoproterenol (0.08 micrograms/kg/min). In the control state, edrophonium administration caused no change in left ventricular dP/dt, fractional shortening or velocity of contraction, but produced significant (P less than .001) decreases in left atrial fractional shortening and contraction velocity. During isoproterenol infusion there were significant increases in dP/dt, the velocity of contraction and fractional shortening in both left atrium and ventricle (P less than .001). Administration of edrophonium during this increase in inotropic state produced significant (P less than .01) decreases in left ventricular dP/dt, fractional shortening and contraction velocity. Moreover, after edrophonium, left atrial fractional shortening and velocity of contraction were decreased to values that were significantly (P less than .001) less than control. Thus, cholinergic stimulation caused selective depression of left atrial systolic function at rest and depression of both left atrial and ventricular function during sympathetic stimulation.

The effects of cocaine on the heart and peripheral circulation were examined in seven mongrel dogs. Hemodynamic variables, in addition to data on ventricular relaxation, mean circulatory filling pressure and arterial compliance, were measured during an intravenous infusion (0.5 mg/kg per min) of cocaine. Holter monitor recordings (6 h) and coronary arteriograms were also obtained. Cocaine increased (p less than 0.01) mean aortic pressure from 72 +/- 5 to 92 +/- 5, left ventricular systolic pressure from 102 +/- 3 to 121 +/- 5, left ventricular end-diastolic pressure from 4.9 +/- 1.3 to 8.2 +/- 1.4 and mean circulatory filling pressure from 7.9 +/- 0.4 to 10.9 +/- 0.5 mm Hg. Cardiac index and stroke volume decreased (p less than 0.01) from 166 +/- 17 to 125 +/- 8 ml/min per kg and from 44 +/- 4 to 29 +/- 3 ml, respectively. Ejection fraction decreased (p less than 0.01) from 61 +/- 1 to 49 +/- 3%. Heart rate, first derivative of left ventricular pressure (dP/dt) and right atrial, mean pulmonary artery and pulmonary artery wedge pressures did not change. The result was a 58% increase in systemic vascular resistance and a 32% decrease in arterial compliance. The pressure gradient for venous return did not change, but resistance to venous return increased 42%. Cocaine prolonged (p less than 0.05) the half-time of left ventricular isovolumic relaxation from 13.4 +/- 0.8 to 16.4 +/- 0.8 ms and the time constant of left ventricular isovolumic relaxation from 19.3 +/- 1.2 to 23.6 +/- 1.1 ms.(ABSTRACT TRUNCATED AT 250 WORDS)